Treatment of diseases and disorders using self-renewing colony forming cells cultured and expanded in vitro

ABSTRACT

The present invention relates to methods and uses of cells for the prevention and treatment of a wide variety of diseases and disorders and the repair and regeneration of tissues and organs using low passage and extensively passaged in vitro cultured, self-renewing, colony forming somatic cells (CF-SC). For example, adult bone marrow-derived somatic cells (ABM-SC), or compositions produced by such cells, are useful alone or in combination with other components for treating, for example, cardiovascular, neurological, integumentary, dermatological, periodontal, and immune mediated diseases, disorders, pathologies, and injuries.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/349,394, filed Jan. 12, 2012, now abandoned, which is a continuationof U.S. patent application Ser. No. 12/140,065 (filed Jun. 16, 2008),now pending; which claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application Nos. 60/929,151 and 60/929,152 (eachfiled Jun. 15, 2007); U.S. Provisional Patent Application No. 60/955,204(filed Aug. 10, 2007); and, U.S. Provisional Patent Application No.60/996,093 (filed on Nov. 1, 2007). Each of the above-referenced PatentApplications are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to the generation and use of invitro cultured self-renewing colony forming somatic cells (CF-SC), andcompositions produced by such cells, for the treatment of a variety ofdiseases and disorders. One example of such CF-SC are adult human bonemarrow-derived somatic cells (hABM-SC).

The present invention also relates to manipulation of CF-SC cellpopulations during cultivation to modulate (i.e., up- or down-regulate)production of various soluble or secreted compositions produced by invitro cultured and expanded self-renewing colony forming cells.

The field of the invention also relates to cell-based andtissue-engineering therapies; particularly, methods of using and/oradministering CF-SC, or compositions produced by such cells, includingadministration via incorporation in, or mixture with, pharmaceuticallyacceptable carriers (such as a pharmaceutically acceptable solution or atransient, permanent, or biodegradable matrix).

Cell Based Therapies

There are two major options in using cell-based therapies to manage andtreat chronic and acute tissue damage in which the overall objective isthe functional and/or cosmetic restoration of damaged tissue. These cellbased therapy options include: 1) Cell Replacement—Use of cells toreplace damaged tissue by establishing long-term engraftment; and 2)Supply Trophic Factors—Use of cells and compositions produced by cells(e.g., growth factors) to stimulate endogenous repair mechanisms throughrelease of factors delivered or produced by cells without long-termengraftment.

The present invention relates to use of cell based therapies withoutrelying on long-term cell engraftment. In particular, the inventionrelates to use of cells, and compositions produced by cells, in thetreatment of various diseases and disorders; particularly thoseinvolving tissues and organs with limited self-renewal capability (suchas, for example, neurological and cardiac tissues and organs).

Cell-based therapeutic options in managing and treating tissue damagealso present the possibility for use of autologous or allogeneic cells.Each of these have certain advantages and disadvantages. Use ofautologous cells involves the following factors or parameters:

-   -   Patient is the donor;    -   Requires manufacture of cell product on a patient-by-patient        basis;    -   Variability in the identity, purity and potency of cell product;        and,    -   Lag time between clinical decision to treat and availability of        cells for transplant.

In contrast, the use of allogeneic cells involves the following factorsor parameters:

-   -   Donor is second party (i.e., donor is not the patient);    -   Risk associated with donor variability;    -   Multiple patients treatable per manufactured batch of cell        product;    -   Increased consistency of identity, purity and potency of cell        product; and,    -   Decreased lag time between clinical decision to treat and        availability of cell product.

The present invention relates primarily to treatments involving use ofallogeneic cells. However, it would also be equally possible to performthese same treatments using autologous cells.

Organ and Tissue Repair

The regenerative potential of certain tissues in the mammalian body hasbeen known for centuries, for example tissues like skin and bone areknown to repair themselves after injury. However, a number of conditionsand diseases of the central nervous system (i.e., brain and spinalcord), peripheral nervous system and heart adversely affect humansbecause of the deficit of regenerative capacity in the effected tissues.These conditions and diseases include, for example, spinal cord injury,amyotrophic lateral sclerosis (ALS), Parkinson's disease, stroke,Huntington's disease, traumatic brain injury, brain tumors, FabryDisease, heart diseases (such as congestive heart failure and myocardialinfarction). Clinical management strategies, for example, frequentlyfocus on the prevention of further damage or injury rather thanreplacement or repair of the damaged tissue (e.g., neurons, glial cells,cardiac muscle); include treatment with exogenous steroids andsynthetic, non-cellular pharmaceutical drugs; and have varying degreesof success which may depend on the continued administration of thesteroid or synthetic drug.

For example, the majority of spinal cord injuries are compressioninjuries with the remaining cases involving complete transection of thespinal cord. Current therapeutic treatments for spinal cord injuryinclude the prevention of additional spinal cord injury by physicallystabilizing the spine through surgical and non-surgical procedures andby inhibiting the inflammatory response with steroidal therapy.

Additionally, aging is a major negative component to nearly every commondisease affecting mammals, and one of the principle features of aging ina degeneration of many tissue including those of skin, bone, eye, brain,liver, kidney, heart, vasculature, muscle, et cetera. Furthermore, thealready limited regenerative capacity of certain tissues of the body isknown to decline with age, tissue maintenance and repair mechanisms inalmost every tissue decline over the course of life.

Thus, there is a need to develop new, improved and effective methods oftreatment for diseases and conditions, in particular, neurological andcardiac diseases and age-related degenerative conditions in humans.

Erythropoiesis

Hematopoietic cells in a healthy human or other mammal do not ordinarilyhave a limited long-term self-renewal capability. However, the potentialfor catastrophic loss of blood (or need otherwise for a supplementalsupply of blood) combined with limited supplies of donor blood, entailsthat methods for enhancing, maintaining, or generating red bloodsupplies in vitro are quite desirable.

Blood is a highly specialized circulating tissue consisting of severaltypes of cells suspended in a fluid medium known as plasma. The cellularconstituents are: red blood cells (erythrocytes), which carryrespiratory gases and give it its red color because they containhemoglobin (an iron-containing protein that binds oxygen in the lungsand transports it to tissues in the body), white blood cells(leukocytes), which fight disease, and platelets (thrombocytes), cellfragments which play an important part in the clotting of the blood.Medical terms related to blood often begin with hemo- or hemato- (BE:haemo- and haemato-) from the Greek word “haima” for “blood.” Bloodcells are produced in the bone marrow; in a process calledhematopoiesis. Blood cells are degraded by the spleen and liver. Healthyerythrocytes have a plasma half-life of 120 days before they aresystematically replaced by new erythrocytes created by the process ofhematopoiesis. Blood transfusion is the most common therapeutic use ofblood. It is usually obtained from human donors. As there are differentblood types, and transfusion of the incorrect blood may cause severecomplications, crossmatching is done to ascertain the correct type istransfused.

A shortage of blood donors and inadequate supplies of red blood cellsfor transfusion is a common problem in treating patients worldwide.Accordingly, there is a need for new, improved and effective methods ofincreasing the availability of red blood cells as this would provide ameans for alleviating at least some of the global shortages in red bloodcell supplies.

Skin

The present invention relates in part to treatment of skin wounds. Thereare currently available a number of different treatments for wounds ofthe skin such as epidermal replacement products, dermal replacementproducts, artificial skin products, and wound dressings. Examples ofsome of these are described briefly below.

Epidermal Replacement Products

According to the manufacturer, EPICEL™ (Genzyme Corp., Cambridge, Mass.)is composed of autologous epidermal cells skin grown from biopsy ofpatients own skin for treatment of burns. Cells are co-cultured withmouse feeder cell line into sheets of autologous epidermis.

According to the manufacturer, MYSKIN™ (CellTran LTD, Sheffield, S1 4DPUnited Kingdom) is a cultured autologous epidermal substitute for thetreatment of burns, ulcers and other non-healing wounds. MYSKIN™contains living cells expanded from the tissue of individual patients.MYSKIN™ comprises a layer of keratinocytes (epidermal cells) on anadvanced polymer-like coating which facilitates the transfer of cellsinto the wound where they can initiate healing. MYSKIN™ uses a medicalgrade silicone substrate layer to support cell delivery, wound coverageand allow exudate management.

According to the manufacturer, EPIDEX™ (Modex Therapeutics Ltd,Lausanne, Switzerland) is an autologous epidermal skin equivalent thatis grown directly from stem and pre-cursor cells derived from hair takendirectly from a patient in a non-surgical procedure.

According to the manufacturer, CELLSPRAY™ (Clinical Cell Culture EuropeLtd, Cambridge CB2 1NL, United Kingdom) is a cultured epithelialautograft suspension that is sprayed onto injured skin in order toprovide a rapid epidermal cover, promote healing and optimize scarquality.

Dermal Replacement Products

According to the manufacturer, INTEGRAT™ Dermal Regeneration Template(Integra LifeSciences Corporation, Plainsboro, N.J.) is a bilayermembrane system for skin replacement. The dermal replacement layer ismade of a porous matrix of fibers of cross-linked bovine tendon collagenand a glycosaminoglycan (chondroitin-6-sulfate) that is manufacturedwith a controlled porosity and defined degradation rate. The temporaryepidermal substitute layer is made of synthetic polysiloxane polymer(silicone) and functions to control moisture loss from the wound. Thecollagen dermal replacement layer serves as a matrix for theinfiltration of fibroblasts, macrophages, lymphocytes, and capillariesderived from the wound bed.

According to the manufacturer, DERMAGRAFT™ (Advanced Biohealing, LaJolla, Calif.) Allogeneic newborn fibroblasts grown on a biodegradablemesh scaffold, indicated for full-thickness diabetic ulcers.

According to the manufacturer, PERMACOL™ (Tissue Science Laboratories,Inc. Andover, Mass. 01810) Permacol™ surgical implant iscollagen-derived from porcine dermis which, when implanted in the humanbody, is non-allergenic and long-lasting.

According to the manufacturer, TRANSCYTE™ (Advanced Biohealing, LaJolla, Calif. 92037) TRANSCYTE™ is a human foreskin-derived fibroblasttemporary skin substitute (allogeneic). The product consists of apolymer membrane and newborn human fibroblast cells cultured underaseptic conditions in vitro on a nylon mesh. Prior to cell growth, thisnylon mesh is coated with porcine dermal collagen and bonded to apolymer membrane (silicone). This membrane provides a transparentsynthetic epidermis when the product is applied to the burn. The humanfibroblast-derived temporary skin substitute provides a temporaryprotective barrier. TRANSCYTE™ is transparent and allows direct visualmonitoring of the wound bed.

According to the manufacturer, RENGRANEX™ Gel (Ortho-McNeilPharmaceutical, Inc.© ETHICON, INC.) is a topical wound care productmade of recombinant PDGF in a gel.

Artificial Skin Products (Epidermal and Dermal Combination Products)

According to the manufacturer, PERMADERM™ (Cambrex Bio ScienceWalkersville, Inc., Walkersville, Md.) PERMADERM™ is constructed fromautologous epidermal and dermal layers of the skin and is indicated forthe treatment of severe burns. The product is reported to be pliable andto grow with the patient.

According to the manufacturer, ORCEL™ (Ortec International, New York,N.Y.) Bilayered construct made from allogeneic epidermal cells andfibroblasts cultured in bovine collagen, indicated for split-thicknessburns. The manufacturer reports no evidence of product-derived DNAdetectable in two human patients treated with product at 2 or 3 weeks,respectively.

According to the manufacturer, APLIGRAF™ (Smith & Nephew, London, WC2N6LA United Kingdom) Allogeneic epidermal cells and fibroblasts culturedin bovine collagen, indicated for venous leg ulcers.

Wound Dressings

According to the manufacturer, 3M™ TEGADERM™ Transparent Film Dressing(3M, St. Paul, Minn.) is a breathable film that provides a bacterial andviral barrier to outside contaminants.

According to the manufacturer, TISSEEL™ VH Fibrin Sealant (Baxter,Deerfield, Ill.) is indicated for use as an adjunct to hemostasis.

SUMMARY OF THE INVENTION

The present invention relates to the production and use of stable cellpopulations and compositions produced thereby. The term “stable cellpopulation” as used herein means an isolated, in vitro cultured, cellpopulation that when introduced into a living mammalian organism (suchas a mouse, rat, human, dog, cow, etc.) does not result in detectableproduction of cells which have differentiated into a specialized celltype or cell types (such as a chondrocyte, adipocyte, osteocyte, etc.)and wherein the cells in the cell population express, or maintain theability to express or the ability to be induced to express, detectablelevels of at least one therapeutically useful composition (such asmembrane bound or soluble TNF-alpha receptor, IL-1R antagonists, IL-18antagonists, compositions shown in Table 1A, 1B, 1C, etc.).

Another characteristic of the stable cell populations of the presentinvention is that the cells do not exhibit ectopic differentiation. Theterm “ectopic” means “in the wrong place” or “out of place”. The term“ectopic” comes from the Greek “ektopis” meaning “displacement” (“ek”,out of +“topos”, place=out of place). For example, an ectopic kidney, isone that is not in the usual location; or, an extrauterine pregnancy isan “ectopic pregnancy”. In the present context, an example of ectopicdifferentiation would be cells that when introduced into cardiac tissue,produce bone tissue-like calcifications and/or ossifications. Thisphenomenon has been demonstrated to occur, for example, when mesenchymalstem cells are injected into cardiac tissue. See, Breitbach et al.,“Potential Risks of Bone Marrow Cell Transplantation Into InfarctedHearts”, Blood, Vol. 110, No. 4 (August 2007).

The present invention relates to the generation and use of expanded, invitro cultured, self-renewing colony forming somatic cells (hereinafterreferred to as “CF-SC”), and products produced by such cells, for thetreatment of a variety of diseases and disorders. Further, the presentinvention also relates to the generation and use of extensivelyexpanded, in vitro cultured, self-renewing colony forming somatic cells(hereinafter referred to as “exCF-SC”), and products produced by suchcells, for the treatment of a variety of diseases and disorders. ExCF-SCare self-renewing colony forming somatic cells (CF-SC) which haveundergone at least about 30, at least about 40, or at least about 50cell population doublings during in vitro cultivation. Hence,self-renewing colony forming somatic cells which have been expanded invitro are hereinafter referred to as “CF-SC” (such that, unlessspecified otherwise, this term encompasses both cell populations whichhave undergone less than about 30 population doublings (e.g., less thanabout 5, less than about 10, less than about 15, less than about 20,less than about 25 population doublings) and also cell populations whichhave undergone more than about 30, more than about 40, or more thanabout 50 populations doublings in vitro). One particular example ofCF-SC are adult human bone marrow-derived somatic cells (hereinafterreferred to as “ABM-SC”). Further, one particular example of exCF-SC areadult human bone marrow-derived somatic cells which have undergone atleast about 30, at least about 40, or at least about 50 cell populationdoublings during in vitro cultivation (hereinafter referred to as“exABM-SC”). Accordingly, the term “ABM-SC”, unless specified otherwise,encompasses both ABM-SC cell populations which have undergone less thanabout 30 population doublings (e.g., less than about 5, less than about10, less than about 15, less than about 20, less than about 25population doublings) and also ABM-SC cell populations which haveundergone more than about 30, more than about 40, or more than about 50populations doublings in vitro).

The term “extensively expanded” as used herein refers to cellpopulations which have undergone at least about 30 or more cellpopulation doublings and wherein the cells are non-senescent, are notimmortalized, and continue to maintain the normal karyotype found in thecell species of origin.

As used herein, the term “substantial capacity for self-renewal” meanshaving the ability to go through numerous cycles of cell divisionresulting in the production of multiple generations of cell progeny(thus, with each cell division, one cell produces two “daughter cells”wherein at least one daughter cell is capable of further cell division).One measure of “substantial capacity for self-renewal” is indicated bythe ability of a cell population to undergo at least about 10, 15, 20,25, 30, 35, 40, 45, 50 or more cell doublings. Another measure of“substantial capacity for self-renewal” is indicated by maintenance ofthe ability of a cell population to re-populate, or approach confluencein, a tissue culture vessel after cell culture passaging (when the sameor similar culture conditions are maintained). Thus, an example of“substantial capacity for self-renewal” is demonstrated when a cellpopulation continues to re-populate a tissue culture vessel in a periodof time of at least about 25%, 50%, 60%, 70%, 80%, 90%, 95% or 100% ofthe time required for such re-population during early cell culturedoublings (such as before a cell population has undergone more thanabout 10 population doublings). Another measure of “substantial capacityfor self-renewal” is maintenance of a consistent rate of populationdoubling time or of a consistent and relatively rapid rate of populationdoubling.

As used herein, the term “substantially no multipotent differentiationcapacity” means cell populations which cannot differentiate intomultiple different types of cells, either in vitro or in vivo. Anexample of cells which do have substantial multipotent differentiationcapacity are hematopoietic stem cells which can differentiate into redblood cells, T-cells, B-cells, platelets, etc. either in vitro or invivo. Another example of cells which do have substantial multipotentdifferentiation capacity are mesenchymal stem cells which candifferentiate, for example, into osteocytes (bone), adipocytes (fat), orchondrocytes (cartilage). In contrast, cells in a cell population whichhave “substantially no multipotent differentiation capacity” cannotdifferentiate into multiple cell types in vitro or when introduced intoan organism or target tissue(s) in vivo. In a preferred embodiment ofthe invention, a cell population with “substantially no multipotentdifferentiation capacity” is one in which at least about 80%, 90%, 95%,98%, 99% or 100% of the cells in the cell population cannot be inducedto detectably differentiate in vitro or in vivo into more than one celltype. A “unipotent” cell or “unipotent progenitor cell” is an example ofa cell which has substantially no multipotent differentiation capacity.

As used herein, “stem cell” means a cell or cells possessing thefollowing two properties: 1) capacity for self-renewal, which is theability to go through numerous cycles of cell division while maintainingthe undifferentiated state; and, 2) differentiation potency, which isthe capacity to change into one or more kinds of mature cell types and,upon such change, no longer undergoing cycles of cell division (forexample, capacity to change into an osteocyte, adipocyte, chondrocyte,etc.). As used herein, differentiation potency means the cells areeither totipotent, pluripotent, multipotent, or unipotent progenitorcells. A “mesenchymal stem cell” is a stem cell of this same definitionbut wherein said cell has been derived or obtained from mesenchymetissue (such as, for example, bone marrow, adipose or cartilage). See,Horwitz et al., “Clarification of the nomenclature for MSC: TheInternational Society for Cellular Therapy position statement”,Cytotherapy, vol. 7, no. 5, pp. 393-395 (2005); and references citedtherein.

As used herein, “totipotent” means cells which can become any type ofcell as may be found during any stage of development in the organism ofthe cells origin. Totipotent cells are typically produced by the firstfew divisions of the fertilized egg (i.e., following fusion of an eggand sperm cell). Thus, totipotent cells can differentiate into embryonicand extraembryonic cell types.

As used herein, “pluripotent” means cells which can differentiate intocells derived from any of the three germ layers (endoderm, mesoderm,ectoderm) found in the organism of the cells origin.

As used herein, “multipotent” means cells which can produce multipletypes (i.e., more than one type) of differentiated cells. A mesenchymalstem cell is an example of a multipotent cell.

As used herein, “unipotent” means cells which can produce only one celltype. Unipotent cells have the property of self-renewal, but can changeinto only one kind of mature cell type.

As used herein, “normal karyotype” means having a genetic compositioncomprising chromosomes of the number and of the structure typicallyfound in, and considered normal for, the species from which the cellsare derived.

As used herein, “connective tissue” is one of the four types of tissueusually referenced in traditional classifications (the others beingepithelial, muscle, and nervous tissue). Connective tissue is involvedin organism and organ structure and support and is usually derived frommesoderm. As used herein, “connective tissue” includes those tissuessometimes referred to as “connective tissue proper”, “specializedconnective tissues”, and “embyronic connective tissue”.

“Connective tissue proper” includes areolar (or loose) connectivetissue, which holds organs and epithelia in place and has a variety ofproteinaceous fibres, including collagen and elastin. Connective tissueproper also includes dense connective tissue (or, fibrous connectivetissue) which forms ligaments and tendons.

“Specialized connective tissue” includes blood, bone, cartilage, adiposeand reticular connective tissue. Reticular connective tissue is anetwork of reticular fibres (fine collagen, type III) that form a softskeleton to support the lymphoid organs (lymph nodes, bone marrow, andspleen)

“Embryonic connective tissue” includes mesenchymal connective tissue andmucous connective tissue. Mesenchyme (also known as embryonic connectivetissue) is the mass of tissue that develops mainly from the mesoderm(the middle layer of the trilaminar germ disc) of an embryo. Viscous inconsistency, mesenchyme contains collagen bundles and fibroblasts.Mesenchyme later differentiates into blood vessels, blood-relatedorgans, and connective tissues. Mucous connective tissue (or mucoustissue) is a type of connective tissue found during fetal development;it is most easily found as a component of Wharton's jelly (a gelatinoussubstance within the umbilical cord which serves to protect and insulatecells in the umbilical cord).

As used herein, “immortalized” refers to a cell or cell line which canundergo an indefinite number of cell doublings in vitro. Immortalizedcells acquire such ability through genetic changes which eliminate orcircumvent the natural limit on a cells ability to continually divide.In contrast, “non-immortalized” cells are eukaryotic cells which, whentaken directly from the organism and cultured in vitro (producing a“primary cell culture”), can undergo a limited number of cell doublingsbefore senescencing (losing ability to divide) and dying. For example,primary cultures of most types of mammalian, non-immortalized cells canusually undergo a relatively defined but reproducibly limited range ofcell doublings (depending on the primary cell type) beforedifferentiating, senescing, or dying.

As used herein “long-term engraftment” means the detectable presence ofdonor cells residing within (or as part of) target tissue to which (orin which) said cells were delivered after more than about 4 weeks fromthe time of administration. “More than about 4 weeks” includes timeperiods of more than about 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, and 24weeks. “More than about 4 weeks” also includes time periods of more thanabout 6 months, 8 months, 10 months, 12 months, 18 months, 24 months, 30months, 36 months, 42 months and 48 months.

The present invention also relates to manipulation of CF-SC and exCF-SCcell populations during cultivation to modulate (i.e., up- ordown-regulate) production of various soluble or secreted compositionsproduced by the in vitro cultured and expanded self-renewing colonyforming cells.

The present invention also relates to extensively expanded cellpopulations which are characterized by loss of ability to differentiateinto bone cells (osteocytes). For example, the present invention relatesto extensively expanded cell populations which are characterized by lossof ability to generate calcium deposits when cultured underosteoinductive conditions, including with or without cultivation in thepresence of the supplemental bone morphogen Noggin (see Example 16).(Mouse and Human Noggin: See, e.g., the U.S. National Center forBiotechnology PubMed Protein Database Accession Nos. NP_(—)032737 andNP_(—)005441 (respectively); see also e.g., Valenzuela, et al.,“Identification of mammalian noggin and its expression in the adultnervous system”, J. Neurosci. 15 (9), 6077-6084 (1995)).

The present invention also relates to extensively expanded cellpopulations characterized by the loss of ability to differentiate intobone cells and/or loss of ability to generate calcium deposits (asdescribed above), but wherein said cell populations continue to secrete,or maintain the ability to secrete or to be induced to secrete, at leastone therapeutically useful composition.

The present invention also relates to cell-based and tissue-engineeringtherapies; particularly, methods of using and/or administering CF-SC andexCF-SC, or compositions produced by such cells, includingadministration via incorporation in, or mixture with, pharmaceuticallyacceptable carriers (such as a pharmaceutically acceptable solution or atransient, permanent, or biodegradable matrix).

The present invention also relates to expanded (i.e., in vitro culturedand passaged) and extensively expanded cell populations which arepreferably negative for expression of the STRO-1 cell surface marker.See, e.g., Stewart et al., “STRO-1, HOP-26 (CD63), CD49a and SB-10(CD166) as markers of primitive human marrow stromal cells and theirmore differentiated progeny: a comparative investigation in vitro” CellTissue Res. 2003 September; 313(3):281-90; and, Dennis et al., “TheSTRO-1+ marrow cell population is multipotential” Cells Tissues Organs.2002; 170(2-3):73-82; and, Oyajobi et al., “Isolation andcharacterization of human clonogenic osteoblast progenitorsimmunoselected from fetal bone marrow stroma using STRO-1 monoclonalantibody”, J Bone Miner Res. 1999 March; 14(3):351-61.

The present invention also relates to manufacture and use ofpharmaceutically acceptable compositions containing CF-SC and exCF-SC(for example, ABM-SC and exABM-SC) with additional structural and/ortherapeutic components. As one example, CF-SC or exCF-SC (for example,ABM-SC or exABM-SC) and collagen may be combined in a pharmaceuticallyacceptable solution to generate compositions in liquid, semi-solid, orsolid-like forms (matrices) for use, for example, in the treatment,repair, and regeneration of skin disorders (e.g., skin wounds such asburns, abrasions, lacerations, ulcers, infections).]

The present invention relates generally to use of self-renewing cells,referred to herein as colony-forming somatic cells (CF-SC) includingextensively passaged colony-forming somatic cells (exCF-SC). Examples ofsuch cells are adult human bone marrow-derived somatic cells (ABM-SC)including extensively passaged adult human bone marrow-derived somaticcells (exABM-SC), for use in treatment of various diseases anddisorders; particularly diseases and disorders involving ischemia,trauma, and/or inflammation (such as, for example, heart failure due toacute myocardial infarction (AMI) and stroke).

Self-renewing colony-forming somatic cells (CF-SC) such as adult humanbone marrow-derived somatic cells (ABM-SC) as used in the presentinvention are prepared as described in U.S. Patent Publication No.20030059414 (U.S. application Ser. No. 09/960,244, filed Sep. 21, 2001)and U.S. Patent Publication No. 20040058412 (U.S. application Ser. No.10/251,685, filed Sep. 20, 2002). Each of these patent applications arehereby incorporated by reference in their entirety. In particular, CF-SCisolated from a source population of cells (such as, for example, frombone marrow, fat, skin, placenta, muscle, umbilical cord blood, orconnective tissue) are cultured under low oxygen conditions (e.g., lessthan atmospheric) and passaged at low cell densities such that the CF-SCmaintain an essentially constant population doubling rate throughnumerous population doublings. After expansion of the CF-SC to anappropriate cell number, the CF-SC may be used to generate thecompositions of the present invention. For example, after expansion ofthe CF-SC in vitro for at least about 30, at least about 40, or at leastabout 50 cell population doublings exCF-SC may be used to generatecompositions of the present invention. In one embodiment CF-SC andexCF-SC, as used in the present invention, are derived from bone marrow(and are referred to herein as ABM-SC and exABM-SC, respectively).

One embodiment of CF-SC and exCF-SC (such as for example, ABM-SC andexABM-SC, respectively), as used in the present invention, is anisolated cell population wherein the cells of the cell populationco-express CD49c and CD90 and wherein the cell population maintains adoubling rate of less than about 30 hours after at least about 30, atleast about 40, or at least about 50 cell population doublings.

Another embodiment of CF-SC and exCF-SC (such as for example, ABM-SC andexABM-SC, respectively), as used in the present invention, is anisolated cell population wherein the cells of the cell populationco-express CD49c, CD90, and one or more cell surface proteins selectedfrom the group consisting of CD44, HLA Class-1 antigen, and β (beta)2-Microglobulin, and wherein the cell population maintains a doublingrate of less than about 30 hours after at least about 30, at least about40, or at least about 50 cell population doublings.

Another embodiment of CF-SC and exCF-SC (such as for example, ABM-SC andexABM-SC, respectively), as used in the present invention, is anisolated cell population wherein the cells of the cell populationco-express CD49c and CD90, but are negative for expression of cellsurface protein CD 10, and wherein the cell population maintains adoubling rate of less than about 30 hour after at least about 30, atleast about 40, or at least about 50 cell population doublings.

Another embodiment of CF-SC and exCF-SC (such as for example, ABM-SC andexABM-SC, respectively), as used in the present invention, is anisolated cell population wherein the cells of the cell populationco-express CD49c, CD90, and one or more cell surface proteins selectedfrom the group consisting of CD44, HLA Class-1 antigen, and β (beta)2-Microglobulin, but are negative for expression of cell surface proteinCD10, and wherein the cell population maintains a doubling rate of lessthan about 30 hours after at least about 30, at least about 40, or atleast about 50 cell population doublings.

Another embodiment of CF-SC and exCF-SC (such as for example, ABM-SC andexABM-SC, respectively), as used in the present invention, is anisolated cell population wherein the cells of the cell populationexpress one or more proteins selected from the group consisting ofsoluble proteins shown in Table 1A, 1B and 1C, and wherein the cellpopulation maintains a doubling rate of less than about 30 hours afterat least about 30, at least about 40, or at least about 50 cellpopulation doublings.

Damaged tissues and organs may result from, for example, disease (e.g.,heritable (genetic) or infectious diseases (such as bacterial, viral,and fungal infections)), physical trauma (such as burns, lacerations,abrasions, compression or invasive tissue and organ injuries), ischemia,aging, toxic chemical exposure, ionizing radiation, and dysregulation ofthe immune system (e.g., autoimmune disorders).

The present invention encompasses the use of CF-SC and exCF-SC (such asfor example, ABM-SC and exABM-SC, respectively), CF-SC and exCF-SCpurified protein fractions, supernatants of CF-SC and exCF-SCconditioned media, and fractions of cell-supernatants derived from CF-SCand exCF-SC conditioned media. In one embodiment of the invention, theabove mentioned components may be combined with, or introduced into,physiologically compatible biodegradable matrices which containadditional components such as collagen and/or fibrin (for example,purified natural or recombinant human, bovine, or porcine collagen orfibrin), and/or polyglycolic acid (PGA), and/or additional structural ortherapeutic compounds. Combination matrices such as these may beadministered to the site of tissue or organ damage to promote, enhance,and/or result in repair and/or regeneration of the damaged tissue ororgan.

Embodiments of the invention include use of CF-SC and exCF-SC (such asfor example, ABM-SC and exABM-SC, respectively), incorporated intopharmaceutically acceptable compositions which may be administered in aliquid, semi-solid, or solid-like state. Embodiments of the inventionmay be administered by methods routinely used by those skilled in therelevant art, such as for example, by topical application, as spray-onor aerosolized compositions, by injection, and implantation.

Use of CF-SC and exCF-SC (such as for example, ABM-SC and exABM-SC,respectively), cells and compositions produced by these cells asdescribed in the present invention for tissue regenerative therapies mayprovide a number of benefits compared to previously described tissueregenerative therapies and products. For example, use of the CF-SC andexCF-SC (such as for example, ABM-SC and exABM-SC, respectively),exABM-SC cells and compositions produced thereby provides a means oftissue regenerative therapy which may exhibit reduced adverse immuneresponses (such as reduced inflammation and T-cell activation; see e.g.,Examples 3A, 3B, 5, 18, and 19. Moreover, since ABM-SC and exABM-SCs areimmunologically silent, subjects do not need to be HLA-matched orpre-conditioned prior to treatment. See, Example 10, Part II; see also,FIG. 17.

The present invention also relates to the use of CF-SC and exCF-SC (suchas expanded and extensively expanded adult human bone marrow-derivedsomatic cells (human ABM-SC and exABM-SC, respectively)), and the cellproducts generated by these cells, for inducing, enhancing, and/ormaintaining hematopoiesis (in particular, for the in vitro generationand production of red blood cells (erythrocytes) from hematopoieticprogenitor cells in a process called erythropoiesis). Thus, anotherembodiment of the invention encompasses the use of such cells and/orcompositions produced by such cells, to induce, enhance, and/or maintainthe generation and production of red blood cells (erythrocytes).

Another example of the field of the invention relates to the preventionand treatment of immune, autoimmune, and inflammatory disorders via useof such cells, cell populations, and compositions produced thereby.

In another example, the present invention provides compositions andmethods for repair and regeneration of wounds of the skin (i.e.,epidermis, dermis, hypodermis); including the manufacture and use ofliquid, semi-solid, and solid-like matrices which incorporate CF-SC andexCF-SC (for example, human ABM-SC and exABM-SC), or products generatedby such cells, and additional structural or therapeutic compounds.

Exemplary Results of Preclinical Studies

In vivo preclinical pharmacology studies have demonstrated thebeneficial effects of ABM-SC in treating myocardial infarction andstroke. For example, in a study investigating the effects ofintra-cardiac injection of hABM-SC in a rat model of myocardialinfarction (in particular, to determine the efficacy of hABM-SC inrestoring cardiac function post-AMI (acute myocardial infarction) andevaluate distribution and disposition of hABM-SC), it was shown thathABM-SC produced a significant improvement in cardiac function andsignificantly reduced fibrosis. Furthermore, the hABM-SC were notobserved to remain in the heart four weeks after cardiac injection, norin any of the peripheral organs examined eight weeks after injection.Additionally, in a study investigating the safety and efficacy ofporcine and human ABM-SC in an AMI model in pigs (in particular, toevaluate the feasibility, safety and efficacy of percutaneous,NOGA™-guided endomyocardial administration of cells through a MYOSTAR™catheter) it was demonstrated that this particular delivery method waswell-tolerated and led to significant improvements in cardiacparameters. Likewise, in a comparison of the method of delivery ofhABM-SC and stroke recovery (in particular, to determine efficacy ofhABM-SC in promoting neuromotor recovery from ischemic stroke) it wasobserved that I.V. or intra-cerebral treatment resulted in significantimprovements in neuromotor activity.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a 2-dimensional SDS PAGE separation (pH 3.5 to 10; 12%polyacrylamide) of proteins secreted by human adult bone marrow-derivedsomatic cells (ABM-SC at about 27 population doublings). Each spot onthe gel represents a separate and distinct protein, ranging in size fromapproximately 5-200 kilodaltons (kDa). The X-axis shows proteinsseparated according to isoelectric point (pH 3.5 to 10). The Y-axisshows proteins separated according to molecular weight (via passagethrough 12% polyacrylamide).

FIG. 2 shows photomicrographs of PC-12 differentiation into neuronsusing nerve growth factor (NGF) and conditioned media derived from humanexABM-SC (at about 43 population doublings). FIG. 2A RPMI-ITS mediumonly. FIG. 2B RPMI-ITS supplemented with NGF. FIG. 2C RPMI-ITSsupplemented with a 1:50 dilution of concentrated control media and NGF.FIG. 2D RPMI-ITS supplemented with a 1:50 dilution of concentratedconditioned media derived from human ABM-SC and NGF. Arrows indicateneurite outgrowth. Extent of neurite outgrowth in panel D issignificantly more robust than that of panel B and C.

FIG. 3 is a graphical representation of inhibition of mitogen-induced Tcell proliferation using human ABM-SC. Lot # RECB801 represents ABM-SCthat have been sub-cultured to about 19 population doublings and Lot #RECB906 represents exABM-SC which have been sub-cultured to about 43population doublings. To stimulate T cell proliferation, cultures wereinoculated with 2.5 or 10 microg/mL Phytohaemagglutinin. Cells were thenharvested after 72 hrs later and stained with CD3-PC7 antibody. Humanmesenchymal stem cells were used as a positive control. (Humanmesenchymal stem cells were obtained from Cambrex Research Bioproducts;now owned by Lonza Group Ltd., Basel, Switzerland).

FIG. 4 shows photomicrographs of pig skin 7 days aftersurgically-induced incisional wounding (left two panels, both woundsfrom experimental group #4). Wound No. 3 treated with allogeneic porcineABM-SC (at about 28 population doublings) shows complete wound closurewith virtually no scar (FIG. 4A). In comparison, Wound No. 4 treatedwith vehicle only reveals a visible scar (FIG. 4B). The graph (rightpanel) represents histomorphometric scoring of tissue sections from bothtreatment groups and shows a statistically significant reduction(p=0.03) in the number of histiocytes in the porcine ABM-SC treatedwounds (statistical significance determined using a two-tailed unpairedT-test); compare, bars for “Histiocytes” PBSG versus pABM-SC treated(FIG. 4C).

FIG. 5A is a graphical representation (top panel) of the extent ofre-epithelialization across the incisional wounds 7 days post-treatment.Wounds treated with porcine ABM-SC (at about 28 population doublings)had a thicker epidermis than those treated with vehicle only. Thephotomicrograph in the lower left panel (FIG. 5B) shows (histologically)complete and anatomically correct repair of the epidermis in the woundstreated with porcine ABM-SC. The photomicrograph in the lower rightpanel (FIG. 5C) shows (histologically) porcine ABM-SC (arrow heads)which appear engrafted, at least transiently, in the hypodermis at this7 day time point.

FIG. 6 is a graphical representation of ABM-SC mediated contraction ofhydrated collagen gel lattices seeded 24 hours after cellreconstitution. Human ABM-SC (at about 27 population doublings) werereconstituted in liquid biodegradable collagen-based media (at 1.8×10⁶cells/mL) and then stored for 24 hours at approximately 4-8° C. Thefollowing day the liquid cell suspension was placed into a culture dishto form a semi-solid collagen lattice. The semi-solid collagen latticeswere maintained in a cell culture incubator to facilitate contractionover the course of three days. Collagen lattices prepared without cellsdid not contract, demonstrating that contraction is dependent upon thepresence of cells.

FIG. 7 is a graphical representation of ABM-SC mediated contraction ofhydrated collagen gel lattices seeded at different cell concentrationsutilizing exABM-SC at about 43 population doublings. The datademonstrate that rate and absolute magnitude of contraction is relatedto cell number. Heat inactivated cells do not contract the gels,demonstrating that this activity is a biophysical event.

FIG. 8 is a graphical representation of ABM-SC mediated secretion ofseveral cytokines and matrix proteases (i.e., IL-6, VEGF, Activin-A,MMP-1, and MMP-2) when cultured for 3 days in hydrated collagen gellattices utilizing exABM-SC at about 43 population doublings.

FIG. 9 shows photomicrographs of human ABM-SC reconstituted inbiodegradable collagen-based media as a liquid (left panel (FIG. 9A) ora semi-solid (right panel FIG. 9B) (utilizing exABM-SC at about 43population doublings). When reconstituted using this formulation, thecell suspension can remain as a liquid at 4° C. for more than 24 hrs.When placed in a culture dish and incubated at 37° C., the cellsuspension will solidify within 1-2 hours, giving rise to a semi-solidstructure than can be physically manipulated.

FIG. 10 shows photomicrographs of a solid-like neotissue formed byculturing human ABM-SC (at about 43 population doublings) reconstitutedin the biodegradable collagen-based media for three days. The upper leftpanel (FIG. 10A) shows the pliability of the tissue when stretched. Theupper right panel (FIG. 10B) shows the general texture of the solid-likeneotissue. The lower panel (FIG. 10C) shows a histological section ofthe tissue stained by Masson's Trichrome, demonstrating the richextracellular matrix synthesized by the ABM-SC. Control gels constructedby the same method, but lacking cells, do not stain blue by this method,demonstrating that the collagen and glycosaminoglycan-rich matrix isproduced by the cells.

FIG. 11 shows an example of the quantities of multiple pro-regenerativecytokines secreted by human ABM-SC with and without TNF-alphastimulation. When sub-cultured, ABM-SC secrete potentially therapeuticconcentrations of several growth factors and cytokines known to augmentangiogenesis, inflammation and wound healing. ABM-SC have been shown toconsistently secrete several cytokines and growth factors in vitro;including proangiogenic factors (e.g., SDF-1 alpha, VEGF, ENA-78 andangiogenin), immunomodulators (e.g., IL-6 and IL-8) and scarinhibitors/wound healing modulators (e.g., MMP-1, MMP-2, MMP-13 andActivin-A). Furthermore, the release of several of these factors ismodulated by tumor necrosis factor alpha (TNF-alpha), a knowninflammatory cytokine released during the course of acute tissue injury.

FIG. 12 shows a model injury-response cascade (inflammation,regeneration, and fibrosis from injury through scar) and examples ofmolecules that can play roles in inflammation, regeneration, andfibrosis.

FIG. 13 shows an example of improved cardiac function results in ratstreated with human ABM-SC. Four weeks after treatment, rats receivingABM-SC demonstrated significantly higher +dp/dt (peak positive rate ofpressure change) values (FIG. 13A). Expressing changes in cardiacfunction over the course of the study by subtracting 0 week +dp/dtvalues from 4 week values (“delta +dp/dt”) demonstrated that whilevehicle treated rats had decreases in cardiac function over the courseof the study (negative delta), animals treated with either cellpreparation showed significant improvement in cardiac function (FIG.13B). Compared to vehicle treated rats, those receiving ABM-SCdemonstrated significantly lower tau values (FIG. 13C), suggestingincreased left ventricular compliance. Tau is the time constant ofisovolumetric left ventricular pressure decay. For peak negative rate ofpressure change (−dp/dt), expressing changes in cardiac function overthe course of the study by subtracting 0 week −dp/dt values from 4 weekvalues (“delta −dp/dt”) demonstrated that while vehicle-treated rats haddecreases in cardiac function over the course of the study (negativedelta), animals treated with cell preparation showed significantimprovement in cardiac function (FIG. 13D). [*p<0.05, **p<0.01 by ANOVA]

FIG. 14 shows reduction of fibrosis and enhanced angiogenesis in a ratmodel myocardial infarct treated with hABM-SC. Semi-quantitative scoringwas used to evaluate changes in infarct size in the hearts of ratsreceiving vehicle or ABM-SC seven days after myocardial infarction.Histopathological analysis, performed approximately 30 days afteradministration of ABM-SC, indicated significant reduction in infarctsize in rats receiving hABM-SC compared to vehicle. According to apreset scale, rats receiving hABM-SC had histological scoresapproximately two points lower than vehicle controls. This figure showsan example of typical infarct size reduction.

FIG. 15 shows results obtained from histological (FIG. 15A(neovascularization) and FIG. 15B (fibrosis)), performed approximately30 days after administration of ABM-SC, measurement of changes in theheart structure of rats receiving vehicle or ABM-SC seven days aftermyocardial infarction.

FIG. 16 shows that allogeneic human ABM-SC and exABM-SC suppressmitogen-induced T-cell proliferation in one-way MLR (mixed lymphocytereaction) assay.

FIG. 17 shows that allogeneic porcine ABM-SC fail to illicit T-cellmediated immune response in a 2-way MLR challenge experiment. A DivisionIndex was calculated for samples collected at baseline and 3 or 30 dayspost-treatment and then challenged in vitro with media, vehicle, pABM-SCor ConA. The average division index from all animals at Day 3 (FIG. 17A)or Day 30 (FIG. 17B) for CD3+ cells which were stimulated with ConA wassignificantly higher than the division index for CD3+ cells from vehicleand pABM-SC treated animals at both pre-treatment and necropsy (*p<0.05).

FIG. 18 shows the changes in cardiac fixed perfusion deficit size inthree patients by comparison of baseline (BL) measurements, withmeasurements obtained 90 days post-treatment with hABM-SC.

FIG. 19 shows the changes in cardiac ejection fractions measured inthree patients by comparison of baseline (BL) measurements withmeasurements obtained 90 days post-treatment with hABM-SC.

FIG. 20 shows examples of quantities of erythropoietic cytokinessecreted in vitro by hABM-SC (i.e., IL-6, Activin-A, VEGF, LIF, IGF-II,SDF-1 and SCF). ABM-SC lots were tested for cytokine secretion usingRAYBIO™ Human Cytokine Antibody Array (RayBiotech, Inc.). Cells werefirst cultured in serum-free Advanced DMEM (GIBCO™) for three days togenerate conditioned medium (CM). The CM was then concentrated usingCENTRICON™ PLUS-20 Centrifugal Filter Units (Millipore) prior toanalysis.

FIG. 21 demonstrates that exABM-SC reduce TNF-α levels in vitro in adose-dependent manner. Human exABM-SC (at about 43 population doublings)were tested for their ability to reduce TNF-α levels when cultured atvarious seeding densities (e.g. 10,000 cells/cm², 20,000 cells/cm², and40,000 cells/cm²). Cells were cultured for 3 days in serum-free AdvancedDMEM (GIBCO™) either alone or supplemented with 10 ng/mL TNF-α. Heatinactivated cells were also included as a negative control.Concentration of TNF is shown on the Y-axis. (Y-axis representsconcentration of substances in media which has been concentrated 100×).

FIGS. 22A and 22B demonstrates that reduction of TNF-α appears to bemediated by the secretion of sTNF-RI and sTNF-RII by exABM-SC (at about43 population doublings). Basal level expression of sTNF-RI occurs inthe absence of a pro-inflammatory inducer (A), while sTNF-RII isdetected at appreciable levels only when first primed with TNF-α (B).These data reveal an inverse relationship between the number of cellsseeded and the levels of both sTNF-RI and sTNF-RII detected, suggestingthat the secreted receptors themselves may be binding to and masking theTNF-α. (Y-axis represents concentration of substances in media which hasbeen concentrated 100×).

FIG. 23 demonstrates that secretion levels of IL-IRA (by exABM-SC atabout 43 population doublings) is dose-dependent. Basal level expressionof IL-IRA occurs in the absence of a pro-inflammatory inducer, but whenprimed when TNF-α, soluble levels increase approximately 10-fold.(Y-axis represents concentration of substances in media which has beenconcentrated 100×).

FIG. 24 shows expression of IL-1 receptor antagonist (IL-1RA) and IL-18binding protein (IL-18BP) by exABM-SC. Human exABM-SC express basallevels of IL-1 receptor antagonist (IL-1RA; FIG. 24A) and IL-18 bindingprotein (IL-18BP; FIG. 24B) even in the absence of an inflammatorysignal such as TNF-alpha.

FIGS. 25A, B, and C show that human ABM-SC reduce levels of TNF-alpha(FIG. 25A) and IL-13 (FIG. 25B) while simultaneously inducing elevatedexpression of IL-2 (FIG. 25C) in a Mixed PBMC reaction assay.(R=Responder PBMC, Self=Mitomycin-C treated PBMC isolated from samedonor as Responder, Stim=Mitomycin-C treated PBMC isolated from adifferent donor.)

FIG. 26 shows a graphical representation of inhibition ofmitogen-induced human peripheral blood mononuclear cell (PBMC)proliferation using human ABM-SC. RECB801 represents a particular lot ofABM-SC that have been sub-cultured to about 19 population doublings and# RECB906 represents a particular lot of ABM-SC that have beensub-cultured to about 43 population doublings. To stimulate PBMCproliferation, cultures were inoculated with 2.5 microg/mLphytohaemagglutinin. After 56 hours in culture, cells were pulsed withThymidine-[Methyl-3H] and at 72 hours isotope incorporation wasquantitated (CPM). Human mesenchymal stem cells were included as apositive control.

DETAILED DESCRIPTION OF THE INVENTION

Typically, a stem cell or other early-stage progenitor cells loseplasticity because the cells have committed to a particulardifferentiation pathway. At the biomolecular level, as this processbegins to occur the cell loses the ability to respond to certainsignaling molecules (e.g., mitogens and morphogens) which wouldotherwise drive the cell to divide or become another cell type. Thus, asa cell begins to differentiate, it leaves the cell cycle (i.e., can nolonger go through mitosis) and enters an irreversible state called GOwherein the cell can no longer divide. Entry into GO is also associatedwith replicative senescence (hallmarks of which include increasedexpression of intracellular proteins p21 and p53). Thus, loss ofplasticity (the ability to differentiate into a variety of cell types)is typically considered a prelude to cellular differentiation orcellular senescence. Furthermore, loss of plasticity is also typicallyassociated with the loss of a cells capacity for continued self-renewal.In contrast, to this typical and traditionally accepted scenario, anunexpected and surprising result of the present invention is that theexCF-SC of the present invention (e.g., exABM-SC) continue to self-renew(including self-renewal at a relatively constant rate) despite loss ofplasticity. Accordingly, one embodiment of the present invention aretherapeutically useful “end-stage cells” with a continued capacity forself-renewal (e.g., cells capable of continued self-renewal andproduction of trophic support factors (or “trophic support cells”)). Inanother embodiment, the exCF-SC and exABM-SC of the present invention donot express significant quantities of p21 and/or p53, wherein a“significant quantity” of said molecules is a quantity which isindicative of cell senescence (wherein senescence may require sufficientexpression levels of p21, p53, and/or other cell cycle regulators).

Additionally, most experts in the field of the present invention wouldexpect a non-hematopoietic stromal-type cell that has lost plasticity tohave limited utility or capability of generating or promotingregeneration of organs and tissues. Thus, another surprising andunexpected result of the present invention, is the ability to generateextensively passaged CF-SC (e.g., ABM-SC) which have lost plasticity yetretain the ability to generate new tissue in vitro and to promoteregeneration of tissue in vivo.

The present invention is drawn, inter alia, to methods of repairing,regenerating, and/or rejuvenating tissues using self-renewing cells,referred to herein as colony-forming somatic cells (CF-SC) (an exampleof which are adult human bone marrow-derived somatic cells (ABM-SC)).Self-renewing colony-forming somatic cells (CF-SC) such as adult humanbone marrow-derived somatic cells (ABM-SC) as used in the presentinvention are prepared as described in U.S. Patent Publication No.20030059414 (U.S. application Ser. No. 09/260,244, filed Sep. 21, 2001)and U.S. Patent Publication No. 20040058412 (U.S. application Ser. No.10/251,685, filed Sep. 20, 2002). Each of these patent applications arehereby incorporated by reference in their entirety. Also incorporated byreference herein are U.S. Provisional Patent Applications 60/929,151 and60/929,152 (each filed on Jun. 15, 2007), U.S. Provisional PatentApplication 60/955,204 (filed on Aug. 10, 2007), and U.S. ProvisionalPatent Application 60/996,093 (filed on Nov. 1, 2007).

In particular, CF-SC isolated from a source population of cells (suchas, for example, from bone marrow (ABM-SC and exABM-SC), fat, skin,placenta, muscle, umbilical cord blood, or connective tissue), arepermitted to adhere to a cell culture surface in the presence of anappropriate media (such as for example, but not limited to, MinimalEssential Medium-Alpha (e.g., available from HYCLONE™) supplemented with4 mM glutamine and 10% fetal bovine serum) and cultured under low oxygenconditions (such as for example, but not limited to, O₂ at about 2-5%,CO₂ at about 5%, balanced with nitrogen) and subsequently passaged atlow cell densities (such as at about 30-1000 cells/cm²) such that theCF-SC maintain an essentially constant population doubling rate (such asfor example, but not limited to, a doubling rate of less than about 30hours) through numerous population doublings (such as for example, butnot limited to, going through 10, 15, 20, 25, 30, 35, 40, 45 and/or 50population doublings).

Embodiments of the invention may be generated with CF-SC and exCF-SC(for example, ABM-SC and exABM-SC) cultured under low oxygen conditionswherein said O₂ concentrations range from about 1-20% (for example,wherein the O₂ concentration is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, or 20%), plus CO₂ and balanced withnitrogen. For example, ABM-SC may be cultured under low oxygenconditions wherein said O₂ concentrations are about 20%, less than about20%, about 15%, less than about 15%, about 10%, less than about 10%,about 7%, less than about 7%, about 6%, less than about 6%, about 5%,less than about 5%, about 4%, less than about 4%, about 3%, less thanabout 3%, about 2%, less than about 2%, about 1%; or, wherein said lowoxygen conditions are in a range from about 1% to about 20%, about 1% toabout 15%, about 1% to about 10%, about 1% to about 5%, about 5% toabout 20%, about 5% to about 15%, about 5% to about 10%, about 10% toabout 15%, about 10% to about 20%, about 2% to about 8%, about 2% toabout 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about4%, about 2% to about 3%, about 3% to about 8%, about 3% to about 7%,about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% toabout 5%, about 5% to about 8%, about 5% to about 7%, about 5% to about6%, or about 5%.

Embodiments of the invention may be generated with CF-SC and exCF-SC(for example, ABM-SC and exABM-SC) cultured under low oxygen conditionswherein CO₂ concentration range from about 1-15% (for example, whereinthe CO₂ concentration is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, or 15%), plus low O₂ and balanced with nitrogen.Embodiments of the invention may be generated with CF-SC and exCF-SC(for example, ABM-SC and exABM-SC) passaged by seeding cells at low celldensities, wherein said cell density ranges from about 1-2500 cells/cm²(for example, wherein the cell density is about 1, 2, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1500, 2000 or 2500 cells/cm²). For example, ABM-SC may be passaged atseeding densities of less than about 2500 cell/cm², less than about 1000cells/cm², less than about 500 cells/cm², less than about 100 cells/cm²,less than about 50 cells/cm², less than about 30 cells/cm², or less thanabout 10 cells/cm². Embodiments of the invention may be generated withCF-SC and exCF-SC (for example, ABM-SC and exABM-SC) wherein the cellpopulation doubling rates are maintained in a range of less than about24-96 hours (for example, wherein the cell population doubling rate ismaintained at less than about 24, 30, 36, 42, 48, 54, 60, 66, 72, 78,84, 90, or 96 hours). Embodiments of the invention may be generated withCF-SC and exCF-SC (for example, ABM-SC and exABM-SC) wherein the cellpopulation maintains an essentially constant doubling rate through arange of population doublings such as in a range of about 5-50population doublings (for example, wherein the population doubling rateis maintained for about 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45,or 5-50 population doublings).

Embodiments of the invention include use of CF-SC and exCF-SC (forexample, ABM-SC and exABM-SC) incorporated into pharmaceuticallyacceptable compositions which may be in a liquid, semi-solid, orsolid-like state. Use of the terms “liquid, semi-solid, or solid-likestate” is intended to indicate that the pharmaceutically acceptablecomposition in which the cells are contained can span a range ofphysical states from 1) a common liquid state (such as in an ordinaryphysiological saline solution); 2) to a wide-range of low-to-highlyviscous states including jelly-like, gelatinous, or viscoelastic states(wherein the pharmaceutical composition contains from very high to verylow levels of extracellular water, for example, such that thecomposition ranges in viscosity from a state where it “oozes” slowlylike oil or honey to increasingly gelatinous or viscoelastic stateswhich may be jelly-like, pliable, semi-elastic and/or malleable; 3) to asolid-like state (having very low levels of extracellular water) whereinthe living cells within the matrix have remodeled the milieu in whichthey were initially suspended into a durable, non-gelatinous, but stillpliable, semi-elastic, and malleable matrix (which, for example, hassome of the same pliable, semi-elastic properties of mammalian skin);see, FIGS. 10A and 10B.

(Note: Viscoelasticity, also known as anelasticity, describes materialsthat exhibit both viscous and elastic characteristics when undergoingplastic deformation. Viscous materials, like honey, resist shear flowand strain linearly with time when a stress is applied. Elasticmaterials strain instantaneously when stretched and just as quicklyreturn to their original state once the stress is removed. Viscoelasticmaterials have elements of both of these properties and, as such,exhibit time dependent strain.)

Clinical administration of cells in liquid, semi-solid, and solid-likevehicles will enable application of treatments that shape to the contourof the wound bed, without trapping unwanted exudate in the wound.

Combining soluble matrix components such as collagens or fibrin withCF-SC and exCF-SC (for example, ABM-SC and exABM-SC) induces the cellpopulation to up-regulate expression of important secreted proteins suchas cytokines and matrix metalloproteinases. Moreover, application ofABM-SC to surgically-induced wounds appears to facilitate wound closureand prevent scarring thereby resulting in minimal scarring (see, Example7).

Additionally, the apparent immunomodulatory properties of CF-SC andexCF-SC (such as, ABM-SC and exABM-SC) (see e.g., Example 4) makecompositions and therapies incorporating these cells attractive for thetreatment of immunological disorders and diseases involving the skin,such as for example, but not limited to, chronic inflammatorydermatoses, psoriasis, lichen planus, lupus erythematosus (LE),graft-versus-host disease (GVHD), and drug eruptions (i.e., adversecutaneous drug reactions).

Secreted proteins and cell-supernatant fractions from CF-SC and exCF-SC(such as, ABM-SC and exABM-SC) conditioned media can be manufacturedfrom serum-free conditions, concentrated and prepared in such manner asto make them suitable for in vivo use. When prepared this way,conditioned serum-free media from ABM-SC has been demonstrated tocontain numerous pro-regenerative cytokines, growth factors, and matrixproteases in therapeutically effective concentrations (see, Table 1A, 1Band 1C). The complex mixture of hundreds of soluble factors produced byABM-SC can be distinguished by 2D SDS PAGE (see, FIG. 1). Individualproteins and other macromolecules can be excised from these gels andidentified using techniques routinely practiced in the art, such as, forexample, MALDI-TOF mass spectrometry (Matrix Assisted Laser DesorptionIonisation-Time Of Flight spectrometry).

Utilizing the methods disclosed (as well as other separation techniquessuch as chromatography or hollow fiber cell culture systems), thedesired proteins or cell supernatant fractions can be isolated,dialyzed, lyophilized and stored as a solid, or reconstituted in anappropriate vehicle for therapeutic administration. In one embodiment,the proteins or cell-supernatant fractions would be reconstituted in asemi-solid collagen or fibrin-based vehicle, and applied topically tothe wound bed.

In addition to products generated by CF-SC and exCF-SC (such as, ABM-SCand exABM-SC), any number and type of pharmaceutically acceptable smallmolecules to large macromolecular compounds (including biologics such aslipids, proteins, and nucleic acids) may be incorporated foradministration with a pharmaceutically acceptable carrier such asbiodegradeable matrices in which CF-SC and exCF-SC (such as, ABM-SC andexABM-SC), or products generated by such cells, are contained. As a verysmall sampling, such additional molecules may include small moleculepharmaceuticals such as anti-inflammatories, antibiotics, vitamins, andminerals (such as calcium) to name but a few categories. Likewise, avery small sampling of biologics may include extracellular matrixproteins, blood plasma coagulation proteins, antibodies, growth factors,chemokines, cytokines, lipids (such as cardiolipin and sphingomyelin),and nucleic acids (such as ribozymes, anti-sense oligonucleotides, orcDNA expression constructs); including therapeutically beneficialvariants and derivatives of such molecules such as various isoforms,fragments, and subunits, as well as substitution, insertion, anddeletion variants. These are mentioned merely by way of example, as itcan be appreciated by those skilled in the art that, in combination withthe teachings provided herein, any number of additional structural ortherapeutically beneficial compounds could be included foradministration with a pharmaceutically acceptable carrier such asbiodegradeable matrices in which CF-SC and exCF-SC (such as, ABM-SC andexABM-SC), or products generated by such cells, are contained.

One embodiment of the invention encompasses a method of stimulatingwound closure in a diabetic patient, such as a diabetic foot or venousleg ulcer, or a post-surgical wound. Stimulation of wound closure may bepromoted by treatment with a pharmaceutical composition of CF-SC andexCF-SC (such as, ABM-SC and exABM-SC), or products generated by suchcells, combined with naturally occurring extracellular matrix and/orblood plasma proteins components such as, for example, purified naturalor recombinant human, bovine, porcine, or recombinant collagens,laminins, fibrinogen, and/or thrombin. The pharmaceutical compositionmay be administered to a mammal, including a human, at the site oftissue damage. In another embodiment, a topically administeredbiodegradable matrix is formed from a mixture of components such aspurified natural or recombinant collagen, fibrinogen, and/or thrombin,combined with allogeneic CF-SC and exCF-SC (such as, ABM-SC andexABM-SC).

In another embodiment of the invention, a pharmaceutical composition ofallogeneic cells and matrix are cultured in vitro for an extended periodof time (such as, for example, but not limited to 1 day to one month orlonger), producing the de novo formation of connective tissue. Inanother embodiment of the invention, the biodegradable matrix is bovinecollagen or poylglycolic acid. In another embodiment, the pharmaceuticalcomposition is cultured in serum-free cell media under conditions ofreduced oxygen tension, for example but not limited to, oxygen tensionequivalent to about 4-5% O₂, 5% CO₂, and balanced with nitrogen.

In one embodiment, the invention encompasses a method of preparing apharmaceutical composition comprising the steps:

(a) preparing a solution comprising soluble collagen, serum-free cellculture media supplemented with glutamine, sodium biocarbonate, andHEPES (optionally including supplementation with insulin, transferrin,and/or selenium);

(b) re-suspending CF-SC or exCF-SC (for example, ABM-SC or exABM-SC) inthe solution; and,

(c) transferring the cell suspension to a tissue mold, or equivalentthereof, to congeal at 37° C., for example, when placed in a cellculture incubator.

The above method of preparing a pharmaceutical composition mayadditionally comprise the step of incubating the culture for an extendedperiod of time (such as, for example but not limited to, 1-3 days orlonger) under low oxygen tension conditions equivalent to about 4-5% O₂,5% CO₂, and balanced with nitrogen.

In another embodiment, the invention encompasses a method of preparing apharmaceutical composition comprising the steps of:

a) preparing a solution comprising fibrinogen and thrombin;

b) re-suspending CF-SC or exCF-SC (for example, ABM-SC or exABM-SC) inthe solution; and,

c) administering the re-suspended solution to an open wound.

In another embodiment, the invention encompasses a method of preparing apharmaceutical composition comprising the steps of:

a) preparing a solution comprising soluble collagen, serum-free cellculture media supplemented with glutamine, sodium biocarbonate, andHEPES (optionally including supplementation with insulin, transferrin,and/or selenium); and

b) mixing a fraction or fractions of cell-supernatant derived from CF-SCor exCF-SC (for example, ABM-SC or exABM-SC) into the solution; and,

c) transferring the solution to a tissue mold, or equivalent thereof, tocongeal at 37° C., for example, when placed in a cell culture incubator.

The above method of preparing a pharmaceutical composition mayadditionally comprise the step of incubating the tissue mold, orequivalent thereof, under atmospheric oxygen tension conditionsequivalent to about 18-21% O₂ and 5% CO₂

In another embodiment, the invention encompasses a method of preparing apharmaceutical composition comprising the steps of:

d) preparing a solution comprising fibrinogen and thrombin;

e) mixing a fraction or fractions of cell-supernatant derived from CF-SCor exCF-SC (for example, ABM-SC or exABM-SC) into the solution; and,

f) administering the solution to an open wound.

In another embodiment, the present invention encompasses tissueregeneration, particularly in the treatment of tissue damage caused byimmune related disorders (such as autoimmune disorders); inflammation(including both acute and chronic inflammatory disorders); ischemia(such as myocardial infarction); traumatic injury (such as burns,lacerations, and abrasions); infection (such as bacterial, viral, andfungal infections); and, chronic cutaneous wounds. The present inventionencompasses treatment of a diversity of damage and disorders, forexample, but not limited to, neurological damage and disorders of thecentral nervous system (brain) and peripheral nervous system (e.g.,spinal cord) (for example, such as may be caused by neurotrauma andneurodegenerative diseases). Another embodiment of the inventionencompasses treatment of diseases and disorders requiring bone,connective tissue, and cartilage regeneration, chronic and acuteinflammatory liver diseases, vascular insufficiency, and corneal andmacular degeneration. Another embodiment of the invention encompassestreating cardiovascular and pulmonary damage and disorders (for example,such as myocardial ischemia and repair and regeneration of bloodvessels). Another embodiment of the invention encompasses treatingdamage and disorders of pancreatic and hepatic tissue as well as otherendocrine and exocrine glands. Another embodiment of the inventionencompasses treating damage and disorders of thymus as well as otherimmune cell producing and harboring organs. Another embodiment of theinvention encompasses treating damage and disorders of the genitourinarysystem (for example, such as the ureter and bladder). Another embodimentof the invention encompasses treating hernias and herniated tissues.Another embodiment of the invention encompasses treatment, repair,regeneration, and reconstruction of heart valves.

CF-SC and exCF-SC (such as, ABM-SC and exABM-SC) or protein andcell-supernatant fractions derived from CF-SC and exCF-SC (such as,ABM-SC and exABM-SC), can also be reconstituted in a solid-likecollagen-base device. When the cells are reconstituted in such manner,the solid-like collagen matrix is remodeled over several days, givingrise to a neotissue that has fabricated its own unique matrix. SuchCF-SC and exCF-SC (such as, ABM-SC and exABM-SC) derived neotissues arepliable, suturable, and bioactive (see e.g., FIGS. 6, 7, and 10A-C).These structures could also be sterilized, chemically cross-linked,freeze-dried, or further processed, rendering the cells non-viable andincapable of further growth.

Such devices may be particularly beneficial in the treatment of burns,including full thickness burn wounds. To rebuild a vascularized woundbed, patients with severe burns are often treated with an artificialdermal replacement after surgical resection of the dead tissue. Afterthe wound bed has healed, these patients are subsequently treated withartificial skin products or applications of epithelial cells in anattempt to re-grow host epidermis.

Compositions, such as described herein, when used in lieu of aconventional artificial dermal products (e.g., DERMAGRAFT™), mayincrease the longevity of subsequently grafted allogeneic skin, byinhibiting or reducing undesirable T-cell mediated immune reactions(see, Example 5). By modulating T-cell mediated immune responses,compositions of the present invention may permit subsequentreapplication of the artificial skin for a durations adequate tostimulate re-growth of the patients own skin.

The above-referenced ABM-SC have been shown to exhibit the followingproperties:

In Vitro

-   -   Secretion of cytokines important in angiogenesis and tissue        repair.    -   Release of factors for prevention and inhibition of scarring and        matrix turnover.    -   Promotion of migration of endothelial cells indicative of        pro-angiogenic activity.

In Vivo

-   -   Significant improvement in outcomes in multiple animal models of        acute myocardial infarction (AMI) and stroke.    -   Effective and well-tolerated intracardiac or intracerebral        delivery of cells.    -   Cells not detectable in tissues eight weeks post-injection.    -   No measurable immune response against cells.

In one embodiment of the invention, a number of pro-regenerativecellular factors secreted by CF-SC and exCF-SC (such as, ABM-SC andexABM-SC) may be used in treatment, repair, regeneration, and/orrejuvenation of damaged tissues and organs (such as, for example,cardiac and neuronal organs and tissues damaged by, for example, heartfailure due to acute myocardial infarction (AMI) or stroke). Theseinclude factors which can be secreted by CF-SC such as ABM-SC as shownin FIG. 11. For example, these factors include, but are not limited to,SDF-1alpha, VEGF, ENA-78, Angiogenin, BDNF, IL-6, IL-8, ALCAM, MMP-2,Activin, MMP-1, MMP-13, MCP-1. See, FIG. 11. Additional factors, such asthose listed in Table 1A, 1B and 1C, may also be secreted by CF-SC andexCF-SC (such as, ABM-SC and exABM-SC). See e.g., Table 1A, 1B and 1C.

Secretion of pro-regenerative factors by CF-SC and exCF-SC (such as,ABM-SC and exABM-SC) may be enhanced or induced by pre-treatment withstimulatory factors (such as, for example, tumor necrosis factor-alpha(TNF-alpha)) to induce the production of conditioned cell culture mediaor to prime the cells before administration of cells to a patient.

Acute ischemia, trauma or inflammation lead to a constellation ofcellular and chemical events in the affected organs and tissues. Seee.g., FIG. 12. In the inflammation phase there occurs a release offactors and an influx of cells to the injured site. In the regenerationphase there occurs a recruitment of circulating cells for the properrepair of functional tissue. And, in the fibrosis phase, there occurs adeposition of fibrotic scars which potentially compromise organfunction. Moreover, a variety of cytokines and other biologicalmolecules play a diversity of roles in each of these processes. Seee.g., FIG. 12.

Use of CF-SC and exCF-SC (such as, ABM-SC and exABM-SC) in the presentinvention includes methods of treating and preventing inflammation,methods of stimulating organ and tissue regeneration while reducingfibrosis (i.e., tissue scarring), and methods of stimulatingangiogenesis via compositions (e.g., cytokines, proteases, extracellularmatrix proteins, etc) produced by stimulated or unstimulated CF-SC andexCF-SC (such as, ABM-SC and exABM-SC).

In another embodiment, CF-SC and exCF-SC may inhibit the biologicalprocess of fibrosis. Fibrosis is a natural byproduct of wound healing,scarring, and inflammation in many human tissues. Fibrosis, also knownas fibrotic scarring, is a significant impediment to regenerating tissuewith optimal function, especially in the heart and central nervoussystem (CNS), because scar tissue displaces cells needed for optimalorgan function. Treatment with cells disclosed herein helps to preventor reduce fibrosis and thereby facilitates the healing of damagedtissue. The fibrosis may be prevented by additive or synergistic effectsof two or more secreted proteins or cell produced compositions,including membrane bound cell-surface molecules. Additionally matrixproteases induced or produced by the administered CF-SC and exCF-SC(such as, ABM-SC and exABM-SC) may play an important part in preventingfibrosis.

In another exemplary use of the present invention, angiogenesis, alsoknown as neovascularization, is increased in a desired tissue.Angiogenesis, or the formation of new blood vessels, is a key componentof regenerative medicine because newly formed tissue must have a bloodsupply, and angiogenesis is crucial if endothelial cells are lost duringdegenerative processes, disease progression, or acute injuries for whichthe present invention is a treatment. Hence, use of CF-SC and exCF-SC(for example, ABM-SC and exABM-SC) or compositions produced by suchcells are useful in stimulating angiogenesis in target tissues andorgans (especially, for example, in damaged cardiac tissue).Angiogenesis is an important component of tissue repair and can operatein conjunction with fibrosis inhibition to optimize healing of damagedtissues.

Another exemplary use of the present invention involves the stimulationof regeneration or rejuvenation processes without the engraftment of theadministered cells. In vivo studies have shown that long term cellengraftment or tissue-specific differentiation of human ABM-SC orexABM-SC are generally not seen, suggesting that the mechanism by whichthese cells incite tissue regeneration is not through cell replacement,but instead through a host response to the cells themselves and/orfactors they produce. This is not surprising, however, given that therole of ABM-SC in bone marrow is to provide structural and trophicsupport. Hence, the present invention includes treatment of damagedtissues and organs wherein the administered CF-SC and exCF-SC (forexample, ABM-SC and exABM-SC) do not exhibit permanent or long-termtissue or organ engraftment. Instead, the therapeutic CF-SC and exCF-SC(for example, ABM-SC and exABM-SC) provide trophic support factors,suppress cell-death, inhibit fibrosis, inhibit inflammation (e.g.,immune cell inflammatory responses), promote extra-cellular matrixremodeling, and/or stimulate angiogenesis without becoming part of therepaired tissue at a significant or currently detectable level.

A further example of the present invention teaches that after a periodof time, the administered cells are not detected anywhere in theexperimental animal, suggesting the administered cells are completelycleared from the body. This suggests that secreted factors play anessential role in the repair of damaged tissue.

In yet another example of the present invention illustrating itsutility, the hABM-SCs disclosed herein come from one donor source. Assuch, these cells will be allogeneic cell transplants in patients whichmight suggest that these transplanted cells could potentially stimulatean adverse immune response. However, surprisingly, we find thattransplanted allogeneic cells disclosed herein actually can suppressmitogen induced T-cell proliferation in vitro and avoid induction of aT-cell-dependent immune response in vivo. A T-cell mediated immuneresponse is a key factor in immune processes that are detrimental tohealing, regenerative, and rejuvenation processes.

As used herein “an effective amount” is an amount sufficient to producedetectable improvement in tissue, organ, or biological system (e.g.,immune system) performance, function, integrity, structure, orcomposition wherein said improvement is indicative of complete orpartial amelioration, restoration, repair, regeneration, or healing ofthe damaged tissue, organ or biological system.

Table 1A, 1B and 1C shows an extensive list of cytokines, growthfactors, soluble receptors, and matrix proteases secreted by humanABM-SC when sub-cultured in serum-free cell culture media. MediaSupernatant Concentrate #1=Advanced DMEM (Gibco™) supplemented with 4 mML-glutamine. Media Supernatant Concentrate #2=RPMI-1640 containing 4 mML-glutamine and HEPES (HyClone) supplemented withInsulin-Transferrin-Selenium-A (Gibco™)

The results demonstrate that numerous trophic factors and solublereceptors important for tissue regeneration and modulation of the immunesystem are produced by ABM-SC at therapeutically relevant levels whencultured under these conditions. Notably, earlier experimentsdemonstrated that supplementation of the base culture medium withinsulin, transferrin, and selenium was required to achieve secretedprotein levels such as those indicated in Table 1A, 1B and 1C.

Immune Disorders

Cells and compositions of the present invention may be used to prevent,treat, and/or ameliorate, inter alia, immune, autoimmune, andinflammatory diseases and disorders. Some examples of such disorders areindicated below; these lists are exemplary only and are not intended tobe comprehensive with respect to all immune, autoimmune, andinflammatory diseases and disorders; nor should the following beconstrued as limiting with respect to pathologies which may be treatedwith the cells and compositions of the present invention.

Example of Some Diseases with a Complete or Partial Autoimmune Etiology:

Acute disseminated encephalomyelitis (ADEM), Addison's disease,Ankylosing spondylitis, Antiphospholipid antibody syndrome (APS),Aplastic anemia, Autoimmune hepatitis, Autoimmune Oophoritis, Celiacdisease, Crohn's disease, Diabetes mellitus type 1, Gestationalpemphigoid, Goodpasture's syndrome, Graves' disease, Guillain-Barrésyndrome (GBS), Hashimoto's disease, Idiopathic thrombocytopenicpurpura, Kawasaki's Disease, Lupus erythematosus, Multiple sclerosis,Myasthenia gravis, Opsoclonus myoclonus syndrome (OMS), Optic neuritis,Ord's thyroiditis, Pemphigus, Pernicious anaemia, Polyarthritis, Primarybiliary cirrhosis, Rheumatoid arthritis, Reiter's syndrome, Sjögren'ssyndrome, Takayasu's arteritis, Temporal arteritis (also known as “giantcell arteritis”), Warm autoimmune hemolytic anemia, and Wegener'sgranulomatosis.

Examples of Some Diseases Suspected of being Linked to Autoimmunity:

Alopecia universalis, Behcet's disease, Chagas' disease, Chronic fatiguesyndrome, Dysautonomia, Endometriosis, Hidradenitis suppurativa,Interstitial cystitis, Lyme disease, Morphea, Neuromyotonia, Narcolepsy,Psoriasis, Sarcoidosis, Scleroderma, Ulcerative colitis, Vitiligo, andVulvodynia.

Examples of Some Immune Hypersensitivity Diseases and Disorders:

Allergic asthma, Allergic conjunctivitis, Allergic rhinitis (“hayfever”), Anaphylaxis, Myasthenia gravis, Angioedema, Arthus reaction,Atopic dermatitis (eczema), Autoimmune hemolytic anemia, AutoimmunePernicious anemia, Coeliac disease, Contact dermatitis (poison ivy rash,Eosinophilia, Erythroblastosis Fetalis, Farmer's Lung (Arthus-typereaction), for example), Goodpasture's syndrome, Graves' disease,Graves' disease, Hashimoto's thyroiditis, Hemolytic disease of thenewborn, Immune complex glomerulonephritis, Immune thrombocytopenia,Myasthenia gravis, Pemphigus, Rheumatic fever, Rheumatoid arthritis,Serum sickness, Subacute bacterial endocarditis, Symptoms of leprosy,Symptoms of malaria, Symptoms of tuberculosis, Systemic lupuserythematosus, Temporal arteritis, Transfusion reactions, Transplantrejection, and Urticaria (hives).

Example of Some Inflammatory Disorders:

allergies, ankylosing spondylitis, arthritis, asthma, autisticenterocolitis, autoimmune diseases, Behcet's disease, chronicinflammation, glomerulonephritis, inflammatory bowel disease (IBD),inflammatory bowel diseases, pelvic inflammatory disease, psoriasis,psoriatic arthritis, reperfusion injury, rheumatoid arthritis,transplant rejection, and vasculitis.

Example of Some Immunodeficiency Disorders:

B cell deficiencies (such as X-linked agammaglobulinemia and SelectiveImmunoglobulin Deficiency), T cell deficiencies (such as DiGeorge'ssyndrome (Thymic aplasia), Chronic mucocutaneous candidiasis, Hyper-IgMsyndrome and, Interleukin-12 receptor deficiency), Combined T cell and Bcell abnormalities (such as Severe Combined Immunodeficiency Disease(SCID), Wiskott-Aldrich syndrome, and Ataxia-telangiectasia), ComplementDeficiencies (such as Hereditary Angioedema or Hereditary angioneuroticedema and Paroxysmal nocturnal hemoglobinuria), Phagocyte deficiencies(such as Leukocyte adhesion deficiency, Chronic Granulomatous Disease(CGD), Chediak-Higashi syndrome, Job's syndrome (Hyper-IgE syndrome),Cyclic neutropenia, Myeloperoxidase deficiency, Glucose-6-phosphatedehydrogenase deficiency, and Interferon-γ deficiency), and CommonVariable Immunodeficiency (CVID), Vici syndrome, and Acquired immunedeficiency syndrome (AIDS).

Embodiments of the Invention

Particular embodiments of the invention include the following:

A1. A method of administering a therapeutically useful amount of abiological composition or compositions to a subject, comprisingadministering to said subject an isolated population of self-renewingcolony forming cells, wherein the cells in said cell population havesubstantially no multipotent differentiation capacity, wherein saidcells have a normal karyotype, and wherein said cells arenon-immortalized.

A2. A method of administering a therapeutically useful amount of abiological composition or compositions to a subject, comprising:

(i) isolating the biological composition or compositions produced by anisolated population of self-renewing colony forming cells; and,

(ii) administering said biological composition or compositions to saidsubject,

wherein the cells in said cell population have substantially nomultipotent differentiation capacity, wherein said cells have a normalkaryotype, and wherein said cells are non-immortalized.

A3. A method of repairing, treating, or promoting regeneration ofdamaged tissue in a subject, comprising administering to said subject aneffective amount of an isolated population of self-renewing colonyforming cells, wherein the cells in said cell population havesubstantially no multipotent differentiation capacity, wherein saidcells have a normal karyotype, and wherein said cells arenon-immortalized.

A4. A method of repairing, treating, or promoting regeneration ofdamaged tissue in a subject, comprising:

(i) isolating the biological composition or compositions produced by anisolated population of self-renewing colony forming cells; and,

(ii) administering said biological composition or compositions to saidsubject,

wherein the cells in said cell population have substantially nomultipotent differentiation capacity, wherein said cells have a normalkaryotype, and wherein said cells are non-immortalized.

A5. A method of treating or reducing inflammation, immune, or autoimmuneactivity in a subject, comprising administering to said subject aneffective amount of an isolated population of self-renewing colonyforming cells, wherein the cells in said cell population havesubstantially no multipotent differentiation capacity, wherein saidcells have a normal karyotype, and wherein said cells arenon-immortalized.

A6. A method of treating or reducing inflammation, immune, or autoimmuneactivity in a subject, comprising:

(i) isolating the biological composition or compositions produced by anisolated population of self-renewing colony forming cells; and,

(ii) administering said biological composition or compositions to saidsubject,

wherein the cells in said cell population have substantially nomultipotent differentiation capacity, wherein said cells have a normalkaryotype, and wherein said cells are non-immortalized.

A7. The method of any of embodiments A1 to A6, wherein prior toadministration, said cell population has been passaged in vitro for anumber of population doublings sufficient to cause the cells in saidpopulation to lose multipotent differentiation capacity.

A8. The method of any one of embodiments A1 to A7, wherein said cellpopulation has unipotent differentiation capacity.

A9. The method of any of embodiments A1 to A8, wherein said cells havesubstantial capacity for self-renewal.

A10. The method of any of embodiments A1 to A9, wherein prior toadministration said cell population has been passaged in vitro for anumber of population doublings while retaining substantial capacity forself-renewal.

A11. The method of any one of embodiments A1 to A10, wherein the cellsin said isolated cell population are not embryonic stem cells.

A12. The method of any one of embodiments A1 to A11, wherein the cellsin said isolated cell population are not stem cells, mesenchymal stemcells, hematopoietic stem cells, multipotent adult progenitor cells(MAPCs), multipotent adult stem cells (MASCs), or fibroblasts.

A13. The method of any one of embodiments A1 to A12, wherein said cellsdo not differentiate into one or more cell types selected from the groupconsisting of:

a) osteocytes; b) adipocytes; and, c) chondrocytes.

A14. The method of any one of embodiments A1 to A13, wherein said cellsdo not deposit detectable levels of calcium following treatment underosteoinductive conditions.

A15. The method of embodiment A14, wherein said osteoinductiveconditions include exposure to exogenously supplied Noggin.

A16. The method of any one of embodiments A1 to A15, wherein the cellsin said isolated cell population are derived from connective tissue.

A17. The method of any one of embodiments A1 to A16, wherein the cellsin said isolated cell population are stromal cells.

A18. The method of any one of embodiments A1 to A17, wherein the cellsin said isolated cell population co-express CD49c and CD90.

A19. The method of any one of embodiments A1 to A18, wherein the cellpopulation maintains an approximately constant doubling rate throughmultiple in vitro cell doublings,

A20. The method of any one of embodiments A1 to A19, wherein said cellsare negative for detectable expression of one or more antigens selectedfrom the group consisting of:

a) CD10; b) STRO-1; and, c) CD106/VCAM-1.

A21. The method of any one of embodiments A1 to A20, wherein said cellsare positive for detectable expression of one or more antigens selectedfrom the group consisting of:

a) CD44; b) HLA Class-1 antigen; and, c) β (beta) 2-Microglobulin,

A22. The method of any one of embodiments A1 to A21, wherein said cellsexpress or secrete detectable quantities of compositions selected fromthe group consisting of:

a) TNF-RI; b) soluble TNF-RI; c) TNF-RII; d) soluble TNF-RII; e) IL-1Rantagonist;

and, f) IL-18 binding protein.

A23. The method of any one of embodiments A1 to A21, wherein said cellsexpress or secrete detectable quantities of compositions selected fromthe group consisting of compositions shown in Table 1A, 1B and 1C.

A24. The method of any one of embodiments A1 to A23, wherein the cellsin said isolated cell population are initially isolated from a tissuesource selected from the group consisting of:

a) bone marrow; b) adipose tissue/fat; c) skin; d) placental; e)umbilical cord; f) tendon; g) ligament; h) muscle fascia; and, i) otherconnective tissues.

A25. The method of embodiment A24, wherein said tissue source is human.

A26. The method of any one of embodiments A1 to A25, wherein said cellpopulation maintains an approximately constant doubling rate through anumber of in vitro cell doublings selected from the group consisting of:

a) 1 to 5 cell doublings; b) 5 to 10 cell doublings; c) 10 to 20 celldoublings; d) 20 to 30 cell doublings; e) 30 to 40 cell doublings; f) 40to 50 cell doublings; g) 1 to 50 cell doublings; h) 5 to 50 celldoublings; i) 10 to 50 cell doublings; j) 20 to 50 cell doublings; k) 30to 50 cell doublings; l) 1 to 10 cell doublings; m) 1 to 20 celldoublings; n) 1 to 30 cell doublings; o) 1 to 40 cell doublings; p) 5 to20 cell doublings; q) 5 to 30 cell doublings; r) 5 to 40 cell doublings;s) 10 to 30 cell doublings; t) 10 to 40 cell doublings; and, u) 20 to 40cell doublings.

A27. The method of any one of embodiments A1 to A26, wherein said cellpopulation has undergone a number of population doublings selected fromthe group consisting of:

a) at least about 10 population doublings; b) at least about 15population doublings; c) at least about 20 population doublings; d) atleast about 25 population doublings; e) at least about 30 populationdoublings; f) at least about 35 population doublings; g) at least about40 population doublings; h) at least about 45 population doublings; and,i) at least about 50 population doublings.

A28. The method of any one of embodiments A1 to A27, wherein saidbiological composition or compositions are bound in or to the cellsurface of said cell populations.

A29. The method of any one of embodiments A1 to A28, wherein saidbiological composition or compositions are secreted into theextracellular environment of said cell populations.

A30. The method of any one of embodiments A1 to A29, wherein saidbiological composition or compositions are one or more moleculesselected from the group consisting of:

a) proteins; b) carbohydrates; c) lipids; d) fatty acids; e) fatty acidderivatives; d) gases; and, e) nucleic acids.

A31. The method of embodiment A30, wherein said proteins are selectedfrom the group consisting of:

a) glycosylated proteins; b) cytokines; c) chemokines; d) lymphokines;e) growth factors; f) trophic factors, g) morphogenetic proteins; and,h) hormones.

A32. The method of embodiment A31, wherein said wherein said biologicalcomposition or compositions bind to and inactivate, or reduce, thebiological activity of molecules selected from the group consisting of:

a) fatty acids; b) fatty acid derivatives; c) receptor molecules; d)cytokines; e) chemokines; f) lymphokines; g) growth factors; h) trophicfactors, i) morphogenetic proteins; and, j) hormones.

A33. The method of embodiment A32, wherein said biological compositionor compositions are soluble receptors that bind cognate ligands selectedfrom the group consisting of:

a) fatty acids; b) fatty acid derivatives; c) receptor molecules; d)cytokines; e) chemokines; f) lymphokines; g) growth factors; h) trophicfactors, i) morphogenetic proteins; and, j) hormones.

A34. The method of any one of embodiments A1 to A33, wherein said cellsare induced to increase expression of one or more biologicalcompositions.

A35. The method of any one of embodiments A1 to A33, wherein said cellsare induced to express one or more biological compositions.

A36. The method of any one of embodiments A1 to A29, wherein said one ormore biological compositions is/are selected from Table 1A, 1B and 1C.

A37. The method of any one of embodiments A1 to A29, wherein said one ormore biological compositions is selected from the group consisting of:

a) TNF-RI; b) soluble TNF-RI; c) TNF-RII; d) soluble TNF-RII; e) IL-1Rantagonist; and, f) IL-18 binding protein.

A38. The method of any one of embodiments A1 to A37, wherein the cellsin said cell population do not exhibit long-term engraftment in, orwith, tissues or organs when administered to a living mammalianorganism.

A39. The method of any one of embodiments A1 to A38, wherein the cellsin said cell population maintain approximately constant levels ofproduction of one or more therapeutically useful compositions in vivo.

A40. The method of embodiment A39, wherein said levels of production aremaintained for a measure of time selected from the group consisting of:

a) at least about 24 hours; b) at least about 48 hours; c) at leastabout 72 hours; d) at least about 4 days; e) at least about 5 days; f)at least about 6 days; g) at least about 7 days; h) at least about 2weeks; i) at least about 3 weeks; j) at least about 4 weeks; k) at leastabout 1 month; l) at least about 2 months; m) at least about 3 months;n) at least about 6 months; and, o) at least about 1 year.

A41. The method of any one of embodiments A1 to A40, wherein saidpatient is human.

A42. The method of any one of embodiments A1 to A41, wherein said methodis used to treat a disease or disorder selected from the groupconsisting of:

a) a neurological disease or disorder; b) a cardiac disease or disorder;c) a skin disease or disorder; d) a skeletal muscle disease or disorder;e) a respiratory disease or disorder; f) a hepatic disease or disorder;g) a renal disease or disorder; h) a genitourinary system disease ordisorder; i) a bladder disease or disorder; j) an endocrine disease ordisorder; k) a hematopoietic disease or disorder; l) a pancreaticdisease or disorder; m) diabetes; n) an ocular disease or disorder; o) aretinal disease or disorder; p) a gastrointestinal disease or disorder;q) a splenic disease or disorder; r) an immunological disease ordisorder; s) an autoimmune disease or disorder; t) an inflammatorydisease or disorder; u) a hyperproliferative disease or disorder; and,v) cancer.

A43. The method of any one of embodiments A1 to A42, wherein said cellsare genetically modified.

A44. The method of embodiment A43, wherein said cells are geneticallymodified by introduction of a recombinant nucleic acid molecule.

A45. A process for making an isolated cell population in any one ofembodiments A1 to A47, wherein said process comprises:

i) obtaining a source population of cells from an organism; and,

ii) culturing said source population of cells in vitro.

B1. A composition comprising a pharmaceutically acceptable mixture ofself-renewing, colony-forming somatic cells (CF-SC), or conditioned cellculture media derived from such cells, and purified naturally occurringor isolated recombinant extracellular matrix or blood plasma proteins.

B2. The composition of embodiment B 1, wherein said CF-SC are derivedfrom bone marrow.

B3. The composition of embodiments B1 or B2, wherein said CF-SC arederived from a human.

B4. The composition of any one of embodiments B1-B3, wherein said CF-SCare derived from an adult mammal, including humans.

B5. The composition of any one of embodiments B1-B4, wherein said CF-SCexpress one or more secreted proteins shown in Table 1A, 1B and 1C.

B6. The composition of any one of embodiment B1-B5, wherein saidextracellular matrix or blood plasma proteins comprise one or morefull-length or alternatively processed isoforms, proteolytic fragments,or subunits of molecules selected from the group consisting of:

a) collagen; b) elastin; c) fibronectin; d) laminin; e) entactin(nidogen); f) hyaluronic acid; g) polyglycolic acid (PGA); h) fibrinogen(Factor I); i) fibrin; j) prothrombin (Factor II); k) thrombin; 1)anti-thrombin; m) Tissue factor Co-factor of VIIa (Factor III); n)Protein C; o) Protein S; p) protein Z; q) Protein Z-related proteaseinhibitor; r) heparin cofactor II; s) Factor V (proaccelerin, labilefactor); t) Factor-VII; u) Factor-VIII; v) Factor-IX; w) Factor-X; x)Factor-XI; y) Factor-XII; z) Factor-XIII; aa) von Willebrand factor; ab)prekallikrein; ac) high molecular weight kininogen; ad) plasminogen; ae)plasmin; af) tissue-plasminogen activator; ag) urokinase; ah)plasminogen activator inhibitor-1; and, ai) plasminogen activatorinhibitor-2.

B7. The composition of any one of embodiments B1-B6, further comprisingpurified naturally occurring or isolated recombinant cytokines orchemokines.

B8. The composition of any one of embodiments B1-B7, wherein saidextracellular matrix, blood plasma proteins, cytokines, and/orchemokines are derived from humans.

B9. The composition of any one of embodiments B1-B8, wherein saidpharmaceutically acceptable mixture forms a semi-solidified orsolidified matrix.

B10. A method of treating damaged tissue with the composition of any oneof embodiments B1-B8, wherein the composition is a liquid.

B11. The method of embodiment B10, wherein the liquid is applied byinjection.

B12. A method of treating damaged tissue with the composition of any oneof embodiments 1-9, wherein the composition is applied as a liquid butthereafter forms a semi-solidified or solidified matrix.

B13. The method of embodiments B10-B12 wherein said tissue is damaged asa result of a condition selected from the group consisting of:

a) disease; b) physical trauma; c) ischemia; d) aging; e) burn; f)bacterial infection; g) viral infection; h) fungal infection; and, i)dysregulation of the immune system.

B14. The method of embodiment B13, wherein the damaged tissue is skin.

B15. A method of using the composition of any one of embodiments B1-B9for facial skin rejuvenation.

B16. A method of using the composition of any one of embodiments B1-B9,wherein said composition inhibits acute inflammation.

C1. A method for treating, repairing, regenerating, or healing a damagedorgan or tissue comprising contacting said damaged organ or tissue withan effective amount of self-renewing colony forming somatic cells orcompositions produced from such cells so as to effect said treatment,repair, regeneration, or healing of the damaged organ or tissue.

C2. The method of embodiment C1, wherein said damaged organ or tissue iscontacted with an effective amount of self-renewing colony formingsomatic cells or compositions produced from such cells by means selectedfrom the group consisting of:

a) injection into the damaged organ or tissue; b) application onto thedamaged organ or tissue; c) injection proximal to the damaged organ ortissue; d) application proximal to the damaged organ or tissue; and, e)intravenous administration.

C3. The method of embodiments C1 or C2, wherein the cells are derivedfrom bone marrow.

C4. The method of any one of embodiments C1-C3, wherein the cells arehuman.

C5. The method of any one of embodiments C1-C4, wherein the cells, orcompositions produced by said cells, inhibit or reduce adverse immuneresponses (such as cell-mediated autoimmunity), fibrosis (scarring)and/or adverse tissue remodeling (for example, ventricular remodeling).

C6. The method of any one of embodiments C1-C5, wherein the cells, orcompositions produced by said cells, control inflammation and/or inhibitacute inflammation.

C7. The method of any one of embodiments C1-C5, wherein the cells, orcompositions produced by said cells, stimulate or enhance angiogenesis.

C8. The method of any one of embodiments C1-C5, wherein said cells donot exhibit significant or detectable levels of permanent or long-termengraftment into said damaged organs or tissues.

C9. The method of any one of embodiments C1-C8, wherein said damagedorgans are selected from the group consisting of heart, brain, andspinal cord.

C10. The method of any one of embodiments C1-C8, wherein said damagedtissue is selected from the group consisting of cardiac tissue, neuronaltissue (including central and peripheral nervous system tissue), andvascular tissue (including major and minor arteries, veins, andcapillaries).

D1. A composition comprising a pharmaceutically acceptable mixture ofself-renewing, colony-forming somatic cells (CF-SC), or conditioned cellculture media derived from such cells, and purified naturally occurringor isolated recombinant extracellular matrix or blood plasma proteins.

D2. The composition of embodiment D1, wherein said CF-SC are derivedfrom bone marrow.

D3. The composition of embodiments D1 or D2, wherein said CF-SC arederived from a human.

D4. The composition of any one of embodiments D1-D3, wherein said CF-SCare derived from an adult mammal, including humans.

D5. The composition of any one of embodiments D1-D4, wherein said CF-SCexpress one or more secreted proteins shown in Table 1A, 1B and 1C.

D6. The composition of any one of embodiment D1-D5, wherein saidextracellular matrix or blood plasma proteins comprise one or morefull-length or alternatively processed isoforms, proteolytic fragments,or subunits of molecules selected from the group consisting of:

a) collagen; b) elastin; c) fibronectin; d) laminin; e) entactin(nidogen); f) hyaluronic acid; g) polyglycolic acid (PGA); h) fibrinogen(Factor I); i) fibrin; j) prothrombin (Factor II); k) thrombin; l)anti-thrombin; m) Tissue factor Co-factor of VIIa (Factor III); n)Protein C; o) Protein S; p) protein Z; q) Protein Z-related proteaseinhibitor; r) heparin cofactor II; s) Factor V (proaccelerin, labilefactor); t) Factor-VII; u) Factor-VIII; v) Factor-IX; w) Factor-X; x)Factor-XI; y) Factor-XII; z) Factor-XIII; aa) von Willebrand factor; ab)prekallikrein; ac) high molecular weight kininogen; ad) plasminogen; ae)plasmin; af) tissue-plasminogen activator; ag) urokinase; ah)plasminogen activator inhibitor-1; and, ai) plasminogen activatorinhibitor-2.

D7. The composition of any one of embodiments D1-D6, further comprisingpurified naturally occurring or isolated recombinant cytokines orchemokines.

D8. The composition of any one of embodiments D1-D7, wherein saidextracellular matrix, blood plasma proteins, cytokines, and/orchemokines are derived from humans.

D9. The composition of any one of embodiments D1-D8, wherein saidpharmaceutically acceptable mixture forms a semi-solidified orsolidified matrix.

D10. A method of treating damaged tissue with the composition of any oneof embodiments D1-D8, wherein the composition is a liquid.

D11. The method of embodiment D10, wherein the liquid is applied byinjection.

D12. A method of treating damaged tissue with the composition of any oneof embodiments D1-D9, wherein the composition is applied as a liquid butthereafter forms a semi-solidified or solidified matrix.

D13. The method of embodiments D10-D12 wherein said tissue is damaged asa result of a condition selected from the group consisting of:

a) disease; b) physical trauma; c) ischemia; d) aging; e) burn; f)bacterial infection; g) viral infection; h) fungal infection; and, i)dysregulation of the immune system.

D14. The method of embodiment D13, wherein the damaged tissue is skin.

D15. A method of using the composition of any one of embodiments D1-D9for facial skin rejuvenation.

D16. A method of using the composition of any one of embodiments D1-D9,wherein said composition inhibits acute inflammation.

D17. A method for treating, repairing, regenerating, or healing adamaged organ or tissue comprising contacting said damaged organ ortissue with an effective amount of self-renewing colony forming somaticcells or compositions produced from such cells so as to effect saidtreatment, repair, regeneration, or healing of the damaged organ ortissue.

D18. The method of embodiment D17, wherein said damaged organ or tissueis contacted with an effective amount of self-renewing colony formingsomatic cells or compositions produced from such cells by means selectedfrom the group consisting of:

a) injection into the damaged organ or tissue; b) application onto thedamaged organ or tissue; c) injection proximal to the damaged organ ortissue; d) application proximal to the damaged organ or tissue; and, e)intravenous administration.

D19. The method of embodiments D17 or D18, wherein the cells are derivedfrom bone marrow.

D20. The method of any one of embodiments D17-D19, wherein the cells arehuman.

D21. The method of any one of embodiments D17-D20, wherein the cells, orcompositions produced by said cells, inhibit or reduce adverse immuneresponses (such as cell-mediated autoimmunity), fibrosis (scarring)and/or adverse tissue remodeling (for example, ventricular remodeling).

D22. The method of any one of embodiments D12-D21, wherein the cells, orcompositions produced by said cells, control inflammation and/or inhibitacute inflammation.

D23. The method of any one of embodiments D17-D21, wherein the cells, orcompositions produced by said cells, stimulate or enhance angiogenesis.

D24. The method of any one of embodiments D17-D21, wherein said cells donot exhibit significant or detectable levels of permanent or long-termengraftment into said damaged organs or tissues.

D25. The method of any one of embodiments D17-D24, wherein said damagedorgans are selected from the group consisting of heart, brain, andspinal cord.

D26. The method of any one of embodiments D17-D24, wherein said damagedtissue is selected from the group consisting of cardiac tissue, neuronaltissue (including central and peripheral nervous system tissue), andvascular tissue (including major and minor arteries, veins, andcapillaries).

D27. A method of inducing, enhancing, and/or maintaining the generationof new red blood cells in vitro.

D28. The method of embodiment D27, wherein said induction, enhancement,or maintenance is achieved by co-cultivation of hematopoietic precursorcells with self-renewing colony forming cells.

D29. The method of embodiment D28, wherein said self-renewing colonyforming cells are human bone marrow-derived somatic cells (hABM-SC).

D30. The method of embodiment D29, wherein said hABM-SC are derived froman adult.

D31. The method of any one of embodiments D27-D29, wherein saidco-cultivation utilizes a semi-permeable barrier to maintain separationof the hematopoietic precursor cells from the self-renewing colonyforming cells while allowing exchange of compositions produced by saidself-renewing colony forming cells across said barrier.

D32. The method of embodiment D27, wherein said induction, enhancement,or maintenance is achieved by co-cultivation of hematopoietic precursorcells with isolated compositions produced by self-renewing colonyforming cells.

D33. The method of embodiment D32, wherein said self-renewing colonyforming cells are human bone marrow-derived somatic cells (hABM-SC).

D34. The method of embodiment D33, wherein said hABM-SC are derived froman adult.

D35. The method of any one of embodiments D32-D34, wherein said isolatedcompositions are lyophilized.

D36. The method of any one of embodiments D32-D34, wherein said isolatedcompositions are cryopreserved.

D37. The method of any one of embodiments D32-D34, wherein said isolatedcompositions are mixed with one or more pharmaceutically acceptablecarriers.

D38. A method of producing, isolating, purifying, and/or packagingcell-derived compositions and/or trophic factors.

D39. A method of producing conditioned media, wherein said mediacontains sera or is sera-free media.

D40. A method of isolating and purifying fractions and/or cell-derivedcompositions from conditioned media, wherein said media contains sera oris sera-free media.

D41. A method of isolating, cryopreserving, and/or expanding CD34+ CordBlood Cells (CBC).

D42. The method of embodiment D41, wherein said CBC are expanded insuspension cultures.

D43. The method of embodiment D41, wherein said CBC are expanded byco-culturing with a feeder layer of self-renewing colony forming cells.

D44. The method of embodiment D43, wherein said self-renewing colonyforming cells are human bone marrow-derived somatic cells (hABM-SC).

D45. A wash solution comprising Balanced Salt Solution with dextrose(BSSD).

D46. The wash solution of embodiment D45 wherein said dextrose is at aconcentration of about 4.5% dextrose.

D47. The wash solution of embodiment D45 or D46, further comprisinghuman serum albumin.

D48. The wash solution of embodiment D47, wherein said human serumalbumin is at a concentration of about 5% human serum album.

D49. A cryopreservation media comprising dimethyl sulfoxide (DMSO) andhuman serum albumin in a Balanced Salt Solution.

D50. The cryopreservation media of embodiment D49, wherein said DMSOconcentration is about 5% and said HSA concentration is about 5%.

E1. An isolated cell population derived from bone marrow, whereingreater than about 91% of the cells of the cell population co-expressCD49c and CD90, and wherein the cell population has a doubling rate ofless than about 30 hours.

E2. The isolated cell population of embodiment E1, wherein the cellpopulation is derived from human bone marrow.

E3. The isolated cell population of embodiments E1 or E2, wherein thecells of the cell population that co-express CD49c and CD90 do notexpress CD34 and/or CD45.

E4. The isolated cell population according to any one of embodiments E1,E2, or E3, wherein the cells of the cell population that co-expressCD49c and CD90 further express at least one cardiac-relatedtranscription factor selected from the group consisting of GATA-4, Irx4,and NRkx2.5.

E5. The isolated cell population according to any one of embodiments E1,E2, or E3, wherein the cells of the cell population that co-expressCD49c and CD90 further express at least one trophic factor selected fromthe group consisting of:

a) Brain-Derived Neurotrophic Factor (BDNF);

b) Cystatin-C;

c) Interleukin-6 (IL-6);

d) Interleukin-7 (IL-7);

e) Interleukin-11 (IL-11);

f) Nerve Growth Factor (NGF);

g) Neurotrophin-3 (NT-3);

h) Macrophage Chemoattractant Protein-1 (MCP-1);

i) Matrix Metalloproteinase-9 (MMP-9);

j) Stem Cell Factor (SCF); and,

k) Vascular Endothelial Growth Factor (VEGF).

E6. The isolated cell population according to any one of embodiments E1,E2, or E3, wherein the cells of the cell population that co-expressCD49c and CD90 further express p21 or p53, and wherein expression of p53is a relative expression of up to about 3000 transcripts of p53 per 10⁶transcripts of an 18s rRNA and expression of p21 is a relativeexpression of up to about 20,000 transcripts of p21 per 10⁶ transcriptsof an 18s rRNA.

E7. The isolated cell population according to any one of embodiments E1,E2, or E3, wherein the isolated cell population has been cultured invitro through a number of population doublings selected from the groupconsisting of:

a) at least about 15 population doublings;

b) at least about 20 population doublings;

c) at least about 25 population doublings;

d) at least about 30 population doublings;

e) at least about 35 population doublings; and,

f) at least about 40 population doublings.

E8. A method of making an isolated cell population derived from bonemarrow, wherein greater than about 91% of the cells of the cellpopulation co-express CD49c and CD90, and wherein the cell populationhas a doubling rate of less than about 30 hours, comprising the stepsof:

a) culturing a source of the cell population under a low oxygencondition or a low oxidative stress condition to produce an adherentcell population; and,

b) culturing the adherent cell population at a seeding density of lessthan about 2500 cells/cm².

E9. The method of embodiment E8, wherein the cell population is derivedfrom human bone marrow.

E10. The method of embodiments E8 or E9, wherein the source of the cellpopulation in embodiment 8, part a) is cultured at an initial seedingdensity selected from the group consisting of:

a) less than about 75000 cells/cm²; and,

b) less than about 50000 cells/cm².

E11. The method of any one of embodiments E8 to E10, wherein theadherent cell population in embodiment 8, part b) is cultured at aseeding density selected from the group consisting of:

a) less than about 2500 cells/cm²;

b) less than about 1000 cells/cm²;

c) less than about 100 cells/cm²;

d) less than about 50 cells/cm²; and,

e) less than about 30 cells/cm².

E12. The method of any one of embodiments E8 to E11, wherein the lowoxygen condition is selected from the group consisting of:

a) between about 1 to 10% oxygen;

b) between about 2 to 7% oxygen;

d) less than about 20% oxygen;

c) less than about 15% oxygen;

d) less than about 10% oxygen;

e) less than about 5% oxygen; and,

f) about 5% oxygen.

E13. The method of any one of embodiments E8 to E12, further includinglysing the red blood cells in a source of the cell population prior toculturing the source of the cell population.

E14. The method of any one of embodiments E8 to E12, further includingselecting a fractionated source of the cell population by passagethrough a density gradient prior to culturing the source of the cellpopulation.

E15. The method of any one of embodiments E8 to E14, wherein the cellsof the cell population that co-express CD49c and CD90, do not expressCD34 and/or CD45.

E16. The method of any one of embodiments E8 to E15, wherein the cellsof the cell population that co-express CD49c and CD90 further express atleast one cardiac-related transcription factor selected from the groupconsisting of GATA-4, Irx4, and NRkx2.5.

E17. The method of any one of embodiments E8 to E15, wherein the cellsof the cell population that co-express CD49c and CD90 further express atleast one trophic factor selected from the group consisting of:

a) Brain-Derived Neurotrophic Factor (BDNF);

b) Cystatin-C;

c) Interleukin-6 (IL-6);

d) Interleukin-7 (IL-7);

e) Interleukin-11 (IL-11);

f) Nerve Growth Factor (NGF);

g) Neurotrophin-3 (NT-3);

h) Macrophage Chemoattractant Protein-1 (MCP-1);

i) Matrix Metalloproteinase-9 (MMP-9);

j) Stem Cell Factor (SCF); and,

k) Vascular Endothelial Growth Factor (VEGF).

E18. The method of any one of embodiments E8 to E15, wherein the cellsof the cell population that co-express CD49c and CD90 further expressp21 or p53, and wherein expression of p53 is a relative expression of upto about 3000 transcripts of p53 per 10⁶ transcripts of an 18s rRNA andexpression of p21 is a relative expression of up to about 20,000transcripts of p21 per 10⁶ transcripts of an 18s rRNA.

E19. The method of any one of embodiments E8 to E15, wherein theisolated cell population has been cultured in vitro through a number ofpopulation doublings selected from the group consisting of:

a) at least about 15 population doublings;

b) at least about 20 population doublings;

c) at least about 25 population doublings;

d) at least about 30 population doublings;

e) at least about 35 population doublings; and,

f) at least about 40 population doublings.

E20. Use of an isolated cell population according to any one ofembodiments E1 to E7 in the manufacture of a medicament for treating ahuman suffering from a condition selected from the group consisting of:

a) a degenerative condition;

b) an acute injury condition;

c) a neurological condition; and,

d) a cardiac condition.

E21. Use of an isolated cell population according to any one ofembodiments E1 to E7 in the manufacture of a medicament for treating ahuman suffering from a degenerative or acute injury condition.

E22. An isolated cell population derived from bone marrow, whereingreater than about 91% of the cells of the cell population co-expressCD49c and CD90, and wherein the cell population has a doubling rate ofless than about 30 hours under a low oxygen condition.

E23. The isolated cell population of embodiment E22, wherein the cellpopulation is derived from human bone marrow.

E24. The isolated cell population of embodiments E22 or E23, wherein thelow oxygen condition is between about 1 to 10% oxygen.

E25. The isolated cell population of embodiment E24, wherein the lowoxygen condition is about 5% oxygen.

E26. The isolated cell population of any one of embodiments E22 to E25,

wherein the cell population is cultured as an adherent cell populationat a seeding density of less than about 2500 cells/cm².

E27. The isolated cell population of any one of embodiments E22 to E25,wherein the seeding density is less than about 1000 cells/cm².

E28. The isolated cell population of any one of embodiments E22 to E25,wherein the seeding density is less than about 100 cells/cm².]

E29. The isolated cell population of any one of embodiments E22 to E25,wherein the seeding density is less than about 50 cells/cm².

E30. The isolated cell population of any one of embodiments E22 to E25,wherein the seeding density is less than about 30 cells/cm².

E31. A method of making an isolated cell population, wherein greaterthan about 91% of the cells of the cell population co-express CD49c andCD90, and wherein the cell population has a doubling rate of less thanabout 30 hours, comprising the steps of:

a) aspirating bone marrow cells from a human;

b) lysing the red blood cell component of the bone marrow aspirate;

c) seeding the non-lysed bone marrow cells in a tissue culturing device;

d) allowing the non-lysed bone marrow cells to adhere to a surface;

e) culturing the adherent cells under a 5% oxygen condition; and

f) passaging the adherent cells at a seeding density of 30 cells/cm².

E32. An isolated cell population obtainable by the method of embodimentE31.

E33. An isolated cell population obtained by the method of embodimentE31.

E34. A method of making an isolated cell population, wherein greaterthan about 91% of the cells of the cell population co-express CD49c andCD90, and wherein the cell population has a doubling rate of less thanabout 30 hours after 30 cell doublings, comprising the steps of:

-   -   a) aspirating bone marrow cells from a human;    -   b) selecting a fractionated source of the cell population by        passage through a density gradient;    -   c) seeding the fractionated cells in a tissue culturing device;    -   d) allowing the fractionated cells to adhere to a surface;    -   e) culturing the adherent cells under a 5% oxygen condition; and    -   f) passaging the adherent cells at a seeding density of 30        cells/cm².

E35. An isolated cell population obtainable by the method of embodimentE34.

E36. An isolated cell population obtained by the method of embodimentE34.

F1. A method of administering a therapeutically useful amount of abiological composition or compositions to an organ, tissue, or subject,comprising administering to said organ, tissue, or subject an isolatedpopulation of bone marrow-derived self-renewing colony-forming somaticcells (CF-SC), wherein said CF-SC do not have multipotentdifferentiation capacity, wherein said CF-SC have a normal karyotype,wherein said CF-SC are non-immortalized, wherein said CF-SC expressCD13, CD44, CD49c, CD90, HLA Class-1 and β (beta) 2-Microglobulin, andwherein said CF-SC do not express CD10, CD34, CD45, CD62L, or CD106.

F2. A method of administering a therapeutically useful amount of abiological composition or compositions to an organ, tissue, or subject,comprising: (a) isolating the biological composition or compositionsproduced by an isolated population of bone marrow-derived self-renewingcolony forming somatic cells (CF-SC); and, (b) administering saidbiological composition or compositions to said organ, tissue, orsubject, wherein said CF-SC do not have multipotent differentiationcapacity, wherein said CF-SC have a normal karyotype, wherein said CF-SCare non-immortalized, wherein said CF-SC express CD13, CD44, CD49c,CD90, HLA Class-1 and β (beta) 2-Microglobulin, and wherein said CF-SCdo not express CD10, CD34, CD45, CD62L, or CD106.

F3. A method of administering a therapeutically useful amount of abiological composition or compositions to an organ, tissue, or subject,comprising administering to said organ, tissue, or subject an isolatedpopulation of bone marrow-derived self-renewing colony-forming somaticcells (CF-SC), wherein said CF-SC do not have multipotentdifferentiation capacity, wherein said CF-SC have a normal karyotype,wherein said CF-SC are non-immortalized, and wherein said CF-SC areobtained from bone marrow by steps comprising: i) incubating bone marrowcells under a low oxygen condition such that said bone marrow cells whenallowed to adhere to a tissue culture-treated surface produce adherentcolony forming units; and, ii) passaging cells in said adherent colonyforming units at low cell seeding densities.

F4. A method of administering a therapeutically useful amount of abiological composition or compositions to an organ, tissue, or subject,comprising: (a) isolating the biological composition or compositionsproduced by an isolated population of bone marrow-derived self-renewingcolony forming somatic cells (CF-SC); and, (b) administering saidbiological composition or compositions to said organ, tissue, orsubject, wherein said CF-SC do not have multipotent differentiationcapacity, wherein said CF-SC have a normal karyotype, wherein said CF-SCare non-immortalized, and wherein said CF-SC are obtained from bonemarrow by steps comprising: i) incubating bone marrow cells under a lowoxygen condition such that said bone marrow cells when allowed to adhereto a tissue culture-treated surface produce adherent colony formingunits; and, ii) passaging cells in said adherent colony forming units atlow cell seeding densities.

F5. A method of preventing tissue damage or of repairing, treating, orpromoting regeneration of damaged tissue, comprising administering to anorgan, tissue, or subject an isolated population of bone marrow-derivedself-renewing colony-forming cells somatic cells (CF-SC), wherein saidCF-SC do not have multipotent differentiation capacity, wherein saidCF-SC have a normal karyotype, wherein said CF-SC are non-immortalized,wherein said CF-SC express CD13, CD44, CD49c, CD90, HLA Class-1 and β(beta) 2-Microglobulin, and wherein said CF-SC do not express CD10,CD34, CD45, CD62L, or CD106.

F6. A method of preventing tissue damage or of repairing, treating, orpromoting regeneration of damaged tissue in an organ, tissue, orsubject, comprising: (a) isolating the biological composition orcompositions produced by an isolated population of bone marrow-derivedself-renewing colony forming somatic cells (CF-SC); and, (b)administering said biological composition or compositions to said organ,tissue, or subject, wherein said CF-SC do not have multipotentdifferentiation capacity, wherein said CF-SC have a normal karyotype,wherein said CF-SC are non-immortalized, wherein said CF-SC expressCD13, CD44, CD49c, CD90, HLA Class-1 and β (beta) 2-Microglobulin, andwherein said CF-SC do not express CD10, CD34, CD45, CD62L, or CD106.

F7. A method of preventing tissue damage or of repairing, treating, orpromoting regeneration of damaged tissue in an organ, tissue, orsubject, comprising administering to said organ, tissue, or subject anisolated population of bone marrow-derived self-renewing colony-formingcells somatic cells (CF-SC), wherein said CF-SC do not have multipotentdifferentiation capacity, wherein said CF-SC have a normal karyotype,wherein said CF-SC are non-immortalized, and wherein said CF-SC areobtained from bone marrow by steps comprising: i) incubating bone marrowcells under a low oxygen condition such that said bone marrow cells whenallowed to adhere to a tissue culture-treated surface produce adherentcolony forming units; and, ii) passaging cells in said adherent colonyforming units at low cell seeding densities.

F8. A method of preventing tissue damage or of repairing, treating, orpromoting regeneration of damaged tissue in an organ, tissue, orsubject, comprising: (a) isolating the biological composition orcompositions produced by an isolated population of bone marrow-derivedself-renewing colony forming somatic cells (CF-SC); and, (b)administering said biological composition or compositions to said organ,tissue, or subject, wherein said CF-SC do not have multipotentdifferentiation capacity, wherein said CF-SC have a normal karyotype,wherein said CF-SC are non-immortalized, and wherein said CF-SC areobtained from bone marrow by steps comprising: i) incubating bone marrowcells under a low oxygen condition such that said bone marrow cells whenallowed to adhere to a tissue culture-treated surface produce adherentcolony forming units; and, ii) passaging cells in said adherent colonyforming units at low cell seeding densities.

F9. A method of treating or reducing inflammation, immune, or autoimmuneactivity in a organ, tissue, or subject, comprising administering tosaid organ, tissue, or subject an isolated population of bonemarrow-derived self-renewing colony-forming cells somatic cells (CF-SC),wherein said CF-SC do not have multipotent differentiation capacity,wherein said CF-SC have a normal karyotype, wherein said CF-SC arenon-immortalized, wherein said CF-SC express CD13, CD44, CD49c, CD90,HLA Class-1 and β (beta) 2-Microglobulin, and wherein said CF-SC do notexpress CD10, CD34, CD45, CD62L, or CD106.

F10. A method of treating or reducing inflammation, immune, orautoimmune activity in an organ, tissue, or subject, comprising: (a)isolating the biological composition or compositions produced by anisolated population of bone marrow-derived self-renewing colony formingsomatic cells (CF-SC); and, (b) administering said biologicalcomposition or compositions to said organ, tissue, or subject, whereinsaid CF-SC do not have multipotent differentiation capacity, whereinsaid CF-SC have a normal karyotype, wherein said CF-SC arenon-immortalized, wherein said CF-SC express CD13, CD44, CD49c, CD90,HLA Class-1 and β (beta) 2-Microglobulin, and wherein said CF-SC do notexpress CD10, CD34, CD45, CD62L, or CD106.

F11. A method of treating or reducing inflammation, immune, orautoimmune activity in an organ, tissue, or subject, comprisingadministering to said organ, tissue, or subject an isolated populationof bone marrow-derived self-renewing colony-forming cells somatic cells(CF-SC), wherein said CF-SC do not have multipotent differentiationcapacity, wherein said CF-SC have a normal karyotype, wherein said CF-SCare non-immortalized, and wherein said CF-SC are obtained from bonemarrow by steps comprising: i) incubating bone marrow cells under a lowoxygen condition such that said bone marrow cells when allowed to adhereto a tissue culture-treated surface produce adherent colony formingunits; and, ii) passaging cells in said adherent colony forming units atlow cell seeding densities.

F12. A method of treating or reducing inflammation, immune, orautoimmune activity in a organ, tissue, or subject, comprising: (a)isolating the biological composition or compositions produced by anisolated population of bone marrow-derived self-renewing colony formingsomatic cells (CF-SC); and, (b) administering said biologicalcomposition or compositions to said organ, tissue, or subject, whereinsaid CF-SC do not have multipotent differentiation capacity, whereinsaid CF-SC have a normal karyotype, wherein said CF-SC arenon-immortalized, and wherein said CF-SC are obtained from bone marrowby steps comprising: i) incubating bone marrow cells under a low oxygencondition such that said bone marrow cells when allowed to adhere to atissue culture-treated surface produce adherent colony forming units;and, ii) passaging cells in said adherent colony forming units at lowcell seeding densities.

F13. The method of any one of embodiments F1, F2, F5, F6, F9, or F10,wherein said CF-SC further express one or more molecules selected fromthe group consisting of: a) CD29; b) CD59; c) CD147; d) CD166; and, e)telomerase and, wherein said CF-SC further do not express one or moremolecules selected from the group consisting of: f) CD11c; g) CD14; h)CD33; i) CD62P; j) CD80; k) STRO-1; l) HLA-Class-II; m) CD178. n) p53;and, o) p21.

F14. The method of any one of embodiments F3, F4, F7, F8, F11 or F12,wherein said low oxygen condition is about 5% oxygen.

F15. The method of any one of embodiments F3, F4, F7, F8, F11 or F12,wherein said low oxygen condition is selected from the group consistingof: a) less than about 20% oxygen; b) less than about 15% oxygen; c)less than about 10% oxygen; d) less than about 5% oxygen; e) betweenabout 1 to 10% oxygen; f) between about 2 to 7% oxygen; g) between about3 to 6% oxygen; h) between about 4 to 6% oxygen; and, i) between about 4to 5% oxygen.

F16. The method of any one of embodiments F3, F4, F7, F8, F11 or F12,wherein said low cell seeding density is less than about 200 cells/cm2.

F17. The method of any one of embodiments F3, F4, F7, F8, F11 or F12,wherein said low cell seeding density is less than about 100 cells/cm2.

F18. The method of any one of embodiments F3, F4, F7, F8, F11 or F12,wherein said low cell seeding density is less than about 50 cells/cm2.

F19. The method of any one of embodiments F3, F4, F7, F8, F11 or F12,wherein said low cell seeding density is less than about 30 cells/cm2.

F20. The method of any one of embodiments F3, F4, F7, F8, F11 or F12,wherein said low cell seeding density is selected from the groupconsisting of: a) less than about 2500 cells/cm2; b) less than about1000 cells/cm2; and, c) less than about 500 cells/cm2.

F21. The method of any one of embodiments F1 to F20, wherein lack ofsaid multipotent differentiation capacity comprises the inability ofsaid CF-SC to differentiate into osteocytes.

F22. The method of any one of embodiments F1 to F20, wherein lack ofsaid multipotent differentiation capacity comprises the inability ofsaid CF-SC to deposit detectable levels of calcium following treatmentof said CF-SC under osteoinductive conditions.

F23. The method of embodiment F22, wherein said treatment comprisesexposure of said CF-SC to media containing one or more componentsselected from the group consisting of: a) dexamethasone; b) ascorbate;and, c) beta glycerophosphate.

F24. The method of embodiment F23, wherein said treatment furthercomprises exposure to Noggin.

F25. The method of any one of embodiments F1 to F20, wherein lack ofsaid multipotent differentiation capacity comprises the inability ofsaid CF-SC to differentiate into chondrocytes.

F26. The method of any one of embodiments F1 to F20, wherein lack ofsaid multipotent differentiation capacity comprises the inability ofsaid CF-SC to differentiate into adipocytes.

F27. The method of any one of embodiments F1 to F20, wherein said CF-SChave unipotent differentiation capacity.

F28. The method of any one of embodiments F1 to F27, wherein said CF-SChave substantial capacity for self-renewal.

F29. The method of any of embodiments F1 to F28, wherein said CF-SCmaintain an approximately constant population doubling rate throughmultiple in vitro cell doublings.

F30. The method of any one of embodiments F1 to F29, wherein said CF-SCare not embryonic stem cells, hematopoietic stem cells, mesenchymal stemcells, multipotent adult progenitor cells (MAPCs), multipotent adultstem cells (MASCs), or fibroblasts.

F31. The method of any one of embodiments F1 to F30, wherein said bonemarrow is human bone marrow.

F32. The method of any one of embodiments F1 to F31, wherein said cellpopulation has undergone a number of population doublings selected fromthe group consisting of: a) at least about 15 population doublings; b)at least about 20 population doublings; c) at least about 25 populationdoublings; d) at least about 30 population doublings; e) at least about35 population doublings; f) at least about 40 population doublings; g)at least about 45 population doublings; and, h) at least about 50population doublings.

F33. The method of any one of embodiments F1 to F32, wherein said organ,tissue, or subject is human.

F34. The method of any one of embodiments F1 to F33, wherein said methodis used to treat or prevent cell, organ, or tissue damage, or diseasesand disorders selected from the group consisting of: a) neurologicaldamage, disease or disorder; b) cardiac damage, disease or disorder; c)skin damage, disease or disorder; d) periodontal damage, disease ordisorder; e) maxillofacialary damage, disease or disorder; f) skeletalmuscle damage, disease or disorder; g) ligament damage, disease ordisorder; h) pulmonary damage, disease or disorder; i) hepatic damage,disease or disorder; j) renal damage, disease or disorder; k)genitourinary system damage, disease or disorder; l) bladder damage,disease or disorder; m) endocrine damage, disease or disorder; n)hematopoietic damage, disease or disorder; o) pancreatic damage, diseaseor disorder; p) diabetes; q) ocular damage, disease or disorder; r)retinal damage, disease or disorder; s) gastrointestinal disease ordisorder; t) splenic damage, disease or disorder; u) immunologicaldamage, disease or disorder; v) autoimmune damage, disease or disorder;w) inflammatory damage, disease or disorder; x) hyperproliferativedamage, disease or disorder; and, y) cancer.

F35. The method of any one of embodiments F5 to F8, wherein said damageis prevented in an organ or tissue during the course of organ or tissuetransplant.

F36. The method of any one of embodiments F1 to F35, wherein said cellsare genetically modified.

F37. The method of embodiment F36, wherein said cells are geneticallymodified by introduction of a recombinant nucleic acid molecule.

F38. A composition comprising a pharmaceutically acceptable mixture ofpurified naturally occurring or isolated recombinant extracellularmatrix or blood plasma proteins and bone marrow-derived self-renewingcolony-forming somatic cells (CF-SC), wherein said CF-SC have a normalkaryotype, wherein said CF-SC are non-immortalized, wherein said CF-SCexpress CD13, CD44, CD49c, CD90, HLA Class-1 and β (beta)2-Microglobulin, and wherein said CF-SC do not express CD10, CD34, CD45,CD62L, or CD106.

F39. A composition comprising a pharmaceutically acceptable mixture ofpurified naturally occurring or isolated recombinant extracellularmatrix or blood plasma proteins and a biological composition orcompositions produced by an isolated population of bone marrow-derivedself-renewing colony forming somatic cells (CF-SC), wherein said CF-SChave a normal karyotype, wherein said CF-SC are non-immortalized,wherein said CF-SC express CD13, CD44, CD49c, CD90, HLA Class-1 and β(beta) 2-Microglobulin, and wherein said CF-SC do not express CD10,CD34, CD45, CD62L, or CD106.

F40. A composition comprising a pharmaceutically acceptable mixture ofpurified naturally occurring or isolated recombinant extracellularmatrix or blood plasma proteins and bone marrow-derived self-renewingcolony-forming somatic cells (CF-SC), wherein said CF-SC have a normalkaryotype, wherein said CF-SC are non-immortalized, and wherein saidCF-SC are obtained from bone marrow by steps comprising: i) incubatingbone marrow cells under a low oxygen condition such that said bonemarrow cells when allowed to adhere to a tissue culture-treated surfaceproduce adherent colony forming units; and, ii) passaging cells in saidadherent colony forming units at low cell seeding densities.

F41. A composition comprising a pharmaceutically acceptable mixture ofpurified naturally occurring or isolated recombinant extracellularmatrix or blood plasma proteins and a biological composition orcompositions produced by an isolated population of bone marrow-derivedself-renewing colony forming somatic cells (CF-SC), wherein said CF-SChave a normal karyotype, wherein said CF-SC are non-immortalized, andwherein said CF-SC are obtained from bone marrow by steps comprising: i)incubating bone marrow cells under a low oxygen condition such that saidbone marrow cells when allowed to adhere to a tissue culture-treatedsurface produce adherent colony forming units; and, ii) passaging cellsin said adherent colony forming units at low cell seeding densities.

F42. The composition of any one of embodiments F38 to F41, wherein saidextracellular matrix or blood plasma proteins comprise one or morefull-length or alternatively processed isoforms, proteolytic fragments,or subunits of molecules selected from the group consisting of: a)collagen; b) elastin; c) fibronectin; d) laminin; e) entactin (nidogen);f) hyaluronic acid; g) polyglycolic acid (PGA) h) fibrinogen (Factor I);i) fibrin; j) prothrombin (Factor II); k) thrombin; l) anti-thrombin; m)Tissue factor Co-factor of VIIa (Factor III); n) Protein C; o) ProteinS; p) protein Z; q) Protein Z-related protease inhibitor; r) heparincofactor II; s) Factor V (proaccelerin, labile factor); t) Factor-VII;u) Factor-VIII; v) Factor-IX; w) Factor-X; x) Factor-XI; y) Factor-XII;z) Factor-XIII; aa) von Willebrand factor; ab) prekallikrein; ac) highmolecular weight kininogen; ad) plasminogen; ae) plasmin; af)tissue-plasminogen activator; ag) urokinase; ah) plasminogen activatorinhibitor-1; and, ai) plasminogen activator inhibitor-2.

F43. The composition of any one of embodiments F38 to F42, furthercomprising purified naturally occurring or isolated recombinantcytokines or chemokines.

F44. The composition of embodiment F38 or F39, wherein said CF-SCfurther express one or more molecules selected from the group consistingof: a) CD29; b) CD59; c) CD147; d) CD166; and, e) telomerase and,wherein said CF-SC further do not express one or more molecules selectedfrom the group consisting of: f) CD11c; g) CD14; h) CD33; i) CD62P; j)CD80; k) STRO-1; l) HLA-Class-II; m) CD178; n) p53; and, o) p21.

F45. The composition of embodiment F40 or F41, wherein said low oxygencondition is about 5% oxygen.

F46. The composition of embodiment F40 or F41, wherein said low oxygencondition is selected from the group consisting of: a) less than about20% oxygen; b) less than about 15% oxygen; c) less than about 10%oxygen; d) less than about 5% oxygen; e) between about 1 to 10% oxygen;f) between about 2 to 7% oxygen; g) between about 3 to 6% oxygen; h)between about 4 to 6% oxygen; and, i) between about 4 to 5% oxygen.

F47. The composition of embodiment F40 or F41, wherein said low cellseeding density is less than about 200 cells/cm2.

F48. The composition of embodiment F40 or F41, wherein said low cellseeding density is less than about 100 cells/cm2.

F49. The composition of embodiment F40 or F41, wherein said low cellseeding density is less than about 50 cells/cm2.

F50. The composition of embodiment F40 or F41, wherein said low cellseeding density is less than about 30 cells/cm2.

F51. The composition of embodiment F40 or F41, wherein said low cellseeding density is selected from the group consisting of: a) less thanabout 2500 cells/cm2; b) less than about 1000 cells/cm2; and, c) lessthan about 500 cells/cm2.

F52. The composition of any one of embodiments F38 to F51, wherein saidCF-SC have unipotent differentiation capacity.

F53. The composition of any one of embodiments F38 to F52, wherein saidCF-SC have substantial capacity for self-renewal.

F54. The composition of any of embodiments F38 to F53, wherein saidCF-SC maintain an approximately constant population doubling ratethrough multiple in vitro cell doublings.

F55. The composition of any one of embodiments F38 to F54, wherein saidCF-SC are not embryonic stem cells, hematopoietic stem cells,mesenchymal stem cells, multipotent adult progenitor cells (MAPCs),multipotent adult stem cells (MASCs), or fibroblasts.

F56. The composition of any one of embodiments F38 to F55, wherein saidbone marrow is human bone marrow.

F57. The composition of any one of embodiments F38 to F56, wherein saidcell population has undergone a number of population doublings selectedfrom the group consisting of: a) at least about 15 population doublings;b) at least about 20 population doublings; c) at least about 25population doublings; d) at least about 30 population doublings; e) atleast about 35 population doublings; f) at least about 40 populationdoublings; g) at least about 45 population doublings; and, h) at leastabout 50 population doublings.

F58. The composition of any one of embodiments F38 to F57, wherein saidorgan, tissue, or subject is human.

F59. The composition of any one of embodiments F38 to F58, wherein saidmethod is used to treat cell, organ, or tissue damage, diseases anddisorders selected from the group consisting of: a) neurological damage,disease or disorder; b) cardiac damage, disease or disorder; c) skindamage, disease or disorder; d) periodontal damage, disease or disorder;e) maxillofacialary damage, disease or disorder order; f) skeletalmuscle damage, disease or disorder; g) ligament damage, disease ordisorder; h) respiratory damage, disease or disorder; i) hepatic damage,disease or disorder; j) renal damage, disease or disorder; k)genitourinary system damage, disease or disorder; l) bladder damage,disease or disorder; m) endocrine damage, disease or disorder; n)hematopoietic damage, disease or disorder; o) pancreatic damage, diseaseor disorder; p) diabetes; q) ocular damage, disease or disorder; r)retinal damage, disease or disorder; s) gastrointestinal disease ordisorder; t) splenic damage, disease or disorder; u) immunologicaldamage, disease or disorder; v) autoimmune damage, disease or disorder;w) inflammatory damage, disease or disorder; x) hyperproliferativedamage, disease or disorder; and, y) cancer.

F60. The composition of any one of embodiments F38 to F59, wherein saidcells are genetically modified.

F61. The composition of embodiment F60, wherein said cells aregenetically modified by introduction of a recombinant nucleic acidmolecule.

F62. The composition of any one of embodiments F38 to F61, wherein saidpharmaceutically acceptable mixture forms a semi-solidified orsolidified matrix.

F63. A method of treating damaged tissue with the composition of any oneof embodiments F38 to F62, wherein the composition is a liquid.

F64. The method of embodiment F63, wherein the liquid is applied byinjection.

F65. A method of treating damaged tissue with the composition of any oneof embodiments F38 to F64, wherein the composition is applied as aliquid but thereafter forms a semi-solidified or solidified matrix.

F66. The method or composition of any one of embodiments F1 to F65,wherein said CF-SC or biological composition or compositions produced bysaid CF-SC are administered by a means selected from the groupconsisting of: a) injection into the damaged organ or tissue; b)application onto the damaged organ or tissue; c) injection proximal tothe damaged organ or tissue; d) application proximal to the damagedorgan or tissue; e) intravenous administration; and, f) organ or tissueperfusion.

F67. The method or composition of any one of embodiments F1 to F66,wherein said CF-SC, or biological composition or compositions producedby said CF-SC, inhibit or reduce one or more biological effects selectedfrom the group consisting of: a) adverse immune responses; b) fibrosis;c) adverse tissue remodeling; d) organ or tissue ischemia; and, e) celldeath.

F68. The method or composition of embodiment F67, wherein said adverseimmune responses are selected from the group consisting of: a)autoimmune responses; b) inflammation; c) immune cell proliferation; d)immune cell activation; e) immune cell degranulation; and, f) T-cellmediated immune responses.

F69. The method or composition of any one of embodiments F1 to F68,wherein said CF-SC, or biological composition or compositions producedby said CF-SC, stimulate or enhance angiogenesis.

F70. A method of inducing, enhancing, and/or maintaining the generationof new red blood cells in vitro or in vivo, wherein said induction,enhancement, or maintenance is achieved by co-cultivation ofhematopoietic precursor cells with bone marrow-derived self-renewingcolony-forming somatic cells (CF-SC), wherein said CF-SC have a normalkaryotype, wherein said CF-SC are non-immortalized, wherein said CF-SCexpress CD13, CD44, CD49c, CD90, HLA Class-1 and β (beta)2-Microglobulin, and wherein said CF-SC do not express CD10, CD34, CD45,CD62L, or CD 106.

F71. A method of inducing, enhancing, and/or maintaining the generationof new red blood cells in vitro or in vivo, wherein said induction,enhancement, or maintenance is achieved by co-cultivation ofhematopoietic precursor cells with a biological composition orcompositions produced by bone marrow-derived self-renewingcolony-forming somatic cells (CF-SC), wherein said CF-SC have a normalkaryotype, wherein said CF-SC are non-immortalized, wherein said CF-SCexpress CD13, CD44, CD49c, CD90, HLA Class-1 and β (beta)2-Microglobulin, and wherein said CF-SC do not express CD10, CD34, CD45,CD62L, or CD106.

F72. The method of embodiment F70 or F71, wherein said CF-SC furtherexpress one or more molecules selected from the group consisting of: a)CD29; b) CD59; c) CD147; d) CD166; and, e) telomerase and, wherein saidCF-SC further do not express one or more molecules selected from thegroup consisting of: f) CD11c; g) CD14; h) CD33; i) CD62P; j) CD80; k)STRO-1; l) HLA-Class-II; m) CD178; n) p53; and, o) p21.

F73. A method of inducing, enhancing, and/or maintaining the generationof new red blood cells in vitro or in vivo, wherein said induction,enhancement, or maintenance is achieved by co-cultivation ofhematopoietic precursor cells with bone marrow-derived self-renewingcolony-forming somatic cells (CF-SC), wherein said CF-SC have a normalkaryotype, wherein said CF-SC are non-immortalized, and wherein saidCF-SC are obtained from bone marrow by steps comprising: i) incubatingbone marrow cells under a low oxygen condition such that said bonemarrow cells when allowed to adhere to a tissue culture-treated surfaceproduce adherent colony forming units; and, ii) passaging cells in saidadherent colony forming units at low cell seeding densities.

F74. A method of inducing, enhancing, and/or maintaining the generationof new red blood cells in vitro or in vivo, wherein said induction,enhancement, or maintenance is achieved by co-cultivation ofhematopoietic precursor cells with a biological composition orcompositions produced by an isolated population of bone marrow-derivedself-renewing colony forming somatic cells (CF-SC), wherein said CF-SChave a normal karyotype, wherein said CF-SC are non-immortalized, andwherein said CF-SC are obtained from bone marrow by steps comprising: i)incubating bone marrow cells under a low oxygen condition such that saidbone marrow cells when allowed to adhere to a tissue culture-treatedsurface produce adherent colony forming units; and, ii) passaging cellsin said adherent colony forming units at low cell seeding densities.

F75. The method of embodiment F73 or F74, wherein said low oxygencondition is about 5% oxygen.

F76. The method of embodiment F73 or F74, wherein said low oxygencondition is selected from the group consisting of: a) less than about20% oxygen; b) less than about 15% oxygen; c) less than about 10%oxygen; d) less than about 5% oxygen; e) between about 1 to 10% oxygen;f) between about 2 to 7% oxygen; g) between about 3 to 6% oxygen; h)between about 4 to 6% oxygen; and, i) between about 4 to 5% oxygen.

F77. The method of embodiment F73 or F74, wherein said low cell seedingdensity is less than about 200 cells/cm2

F78. The method of embodiment F73 or F74, wherein said low cell seedingdensity is less than about 100 cells/cm2.

F79. The method of embodiment F73 or F74, wherein said low cell seedingdensity is less than about 50 cells/cm2.

F80. The method of embodiment F73 or F74, wherein said low cell seedingdensity is less than about 30 cells/cm2.

F81. The method of embodiment F73 or F74, wherein said low cell seedingdensity is selected from the group consisting of: a) less than about2500 cells/cm2; b) less than about 1000 cells/cm2; and, c) less thanabout 500 cells/cm2.

F82. The method of any one of embodiments F70 to F81, wherein said CF-SChave unipotent differentiation capacity.

F83. The method of any one of embodiments F70 to F82, wherein said CF-SChave substantial capacity for self-renewal.

F84. The method of any one of embodiments F70 to F83, wherein said CF-SCmaintain an approximately constant population doubling rate throughmultiple in vitro cell doublings.

F85. The method of any one of embodiments F70 to F84, wherein said CF-SCare not embryonic stem cells, hematopoietic stem cells, mesenchymal stemcells, multipotent adult progenitor cells (MAPCs), multipotent adultstem cells (MASCs), or fibroblasts.

F86. The method of any one of embodiments F70 to F85, wherein said bonemarrow is human bone marrow.

F87. The method of any one of embodiments F70 to F86, wherein said cellpopulation has undergone a number of population doublings selected fromthe group consisting of: a) at least about 15 population doublings; b)at least about 20 population doublings; c) at least about 25 populationdoublings; d) at least about 30 population doublings; e) at least about35 population doublings; f) at least about 40 population doublings; g)at least about 45 population doublings; and, h) at least about 50population doublings.

F88. The method of any one of embodiments F70 to F87, wherein said cellpopulation is human.

F89. The method of any one of embodiments F9 to F12, wherein saidtreatment or reduction of inflammation, immune, or autoimmune activityresults in one or more changes selected from the group consisting of: a)decreased focal or systemic levels of IL-13; b) decreased focal orsystemic levels of TNF-alpha; c) increased focal or systemic levels ofIL-2; d) decreased focal or systemic immune cell proliferation; e)decreased focal or systemic immune cell activation; and, f) decreasedfocal or systemic immune cell degranulation.

F90. The method of embodiment F89, wherein said decreased immune cellproliferation comprises inhibition of proliferation of one or more celltypes selected from the group consisting of: a) monocytes; b)granulocytes; c) lymphocytes; and, d) neutrophils.

F91. The method of embodiment F89, wherein said increased focal orsystemic levels of IL-2 supports maturation of T-cell regulatory cells.

F92. The method or composition of any one of embodiments F1-F91, whereinsaid biological composition or compositions are lyophilized.

F93. The method or composition of any one of embodiments F1-F91, whereinsaid biological composition or compositions are cryopreserved.

F94. The method or composition of any one of embodiments F1-F91, whereinsaid biological composition or compositions are mixed with one or morepharmaceutically acceptable carriers.

EXAMPLES Example 1 Bioactivity of Adult Bone Marrow-Derived SomaticCells: Production of Serum-Free Conditioned Media

Production of serum-free conditioned media was produced as describedbelow for use in assays, such as the solid-phase antibody capture ofsecreted proteins (also as described below). Human exABM-SC (Lot #RECB-819; at ˜43 population doublings) were thawed and re-suspended ineither Advanced DMEM (GIBCO™; Catalog #12491-015, Lot #1216032(Invitrogen Corp., Carlsbad, Calif., USA)) supplemented with 4 mML-glutamine (Catalog #SH30034.01. Lot #134-7944, (HYCLONE™ LaboratoriesInc., Logan, Utah, USA)) or HyQ® RPMI-1640 (HYCLONE™ Catalog#SH30255.01, Lot # ARC25868) containing 4 mM L-glutamine andsupplemented with Insulin-Transferrin-Selenium-A (ITS) (GIBCO™; Catalog#51300-044, Lot #1349264). Cell suspensions were then seeded in T-225cm² CELLBIND™ (Corning Inc., NY, USA) culture flasks (culture surfacestreated with a patented microwave plasma process; see, U.S. Pat. No.6,617,152) (n=3) at 20,000 cells/cm² in 36 mL of media (n=3 percondition). Cultures were placed in a 37° C. humidified trigas incubator(4% O₂, 5% CO₂, balanced with nitrogen) for approximately 24 hours.Cultures were then re-fed with fresh media on same day to removenon-adherent debris and returned to the incubator. On day 3, cellculture media were concentrated using 20 mL CENTRICON™ PLUS-20Centrifugal Filter Units (Millipore Corp., Billerica, Mass., USA), asper manufacturer's instructions. Briefly, concentrators were centrifugedfor 45 minutes at 1140×G. Concentrated supernatants were transferred toclean 2 mL cryovials and stored at −80° C. Fresh culture media were alsoconcentrated as described for use as a negative control. The cells werethen removed from the flasks using 0.25% porcine trypsin EDTA (CELLGRO™;Catalog #30-004-C1 (Mediatech Inc., Herndon, Va., USA)). Trypsin wasthen neutralized by adding back an equal volume of cell culture mediacontaining 10% fetal bovine serum. Cell count and viability analysis wasperformed using a COULTER™ AcT 10 Series Analyzer (Beckman Coulter,Fullerton, Calif.) and trypan blue exclusion assays, respectively.

To perform 2D SDS-PAGE, human ABM-SC (Lot # PCH627; at ˜27 populationdoublings) were thawed and re-suspended in either HyQ® Minimum EssentialMedium (MEM), Alpha Modification (HYCLONE™; Catalog #SH30265.01, Lot #ASA28110) supplemented with 4 mM L-glutamine (HYCLONE™; Catalog#SH30034.01, Lot #134-7944)) or RPMI1640 (HYCLONE™; Catalog #SH30255.01) supplemented with 4 mM L-glutamine (HYCLONE™; Catalog #SH30034.01, Lot #134-7944). Cell suspensions were then seeded in T-225cm² CELLBIND™ culture flasks (n=3) at 24-40,000 cells/cm² in 36 mL ofmedia (n=3 per condition). Cultures were placed in a 37° C. humidifiedtrigas incubator (4% O₂, 5% CO₂, balanced with nitrogen) forapproximately 24 hours. Cultures were re-fed with fresh media on sameday to remove non-adherent debris and then returned to the incubator.The following day, conditioned media were collected, pooled, andcentrifuged at 1140×G for 15 minutes to remove cell debris, and thentransferred to sterile centrifuge tubes for short-term storage at −80°C.

Example 2 Two Dimensional (2-D) SDS PAGE Separation of Secreted Factors(FIG. 1)

Frozen aliquots of conditioned media and control media (samples) wereshipped to Kendrick Labs, Inc. (Madison, Wis.) for analysis. Prior touse, samples were thawed and warmed to room temperature. Approximately50 mL of each sample was lyophilized then re-dissolved in 200 microL ofSDS Boiling Buffer (5% sodium dodecyl sulfate, 5% beta mercaptoethanolethanol, 10% glycerol and 60 mM Tris, pH 6.8) and 2 mL of ultrapurewater. The samples were then dialyzed against 5 mM Tris, pH 7.0 for twodays at 4° C. using 6-8,000 MWCO membranes. The final dialysis wasperformed using water only. The samples were lyophilized once again,re-dissolved in 200 microL of SDS Boiling Buffer, and heated in aboiling water bath for 5 minutes before loading into the gels.

Two-dimensional gel electrophoresis was performed according to themethod of O'Farrell (O'Farrell, P. H., J. Biol. Chem. 250: 4007-4021,1975) as follows: Isoelectric focusing was first carried out in glasstubes of inner diameter 2.0 mm using 2.0% ampholines, pH 3.5-10(Amersham Biosciences, Piscataway, N.J.) for 20,000 volt-hrs. 50 ng ofIEF internal standard (tropomyosin) was then added to each sample. Thetropomyosin standard is used as a reference point on the gel, itmigrates as a doublet with a lower polypeptide spot of MW 33,000 and pI5.2. The tube gel pH gradient for this set of ampholines was determinedusing a surface pH electrode.

After equilibration for 10 min in buffer 0 (10% glycerol, 50 mmdithiothreitol, 2.3% SDS, 0.0625 M tris, pH 6.8) each tube gel wassealed to the top of a stacking gel that, itself, is placed on top of a12% acrylamide slab gel (1.0 mm thickness). SDS slab gel electrophoresiswas carried out for about 5 hours at 25 mA. The following proteins(Sigma Chemical Co.) were added as molecular weight standards to asingle well in the agarose portion of the gel (the agarose is castbetween the tube gel to the slab gel): myosin (220,000 daltons),phosphorylase A (94,000 daltons), catalase (60,000 daltons), actin(43,000 daltons), carbonic anhydrase (29,000 daltons), and lysozyme(14,000 daltons). Following silver-staining the standards appear asbands on the basic edge of the acrylamide slab gel (Oakley et al. Anal.Biochem. 105:361-363, 1980). The gel was then dried between two sheetsof cellophane paper with the acid end to the left (FIG. 1). If gels areintended for use with mass spectroscopy analysis they are stained usingthe silver stain method of O'Connell and Stults (O'Connell and Stults.Electrophoresis. 18:349-359, 1997).

The results show that using the methods provided, human ABM-SC can becultured in the absence of animal serum to produce conditioned mediarich in secreted proteins, and that such proteins can be individuallyidentified and isolated. Conditioned media produced in such can also beprocessed, alternatively, by fractionating the expressed proteins basedon a range of molecular weights. Techniques for protein concentrationand fractionation are well-known and routinely used by those of ordinaryskill in the art. These techniques include techniques such as affinitychromatography, hollow fiber filtration, 2D PAGE, and low-absorptionultrafiltration.

Example 3A Pro-Regenerative Cytokine Secretion by Human ABM-SC

Human ABM-SC were plated in triplicate at 6,000 viable cells/cm² in cellculture “T” flasks containing AFG104 media. After allowing cells toattach and equilibrate for 24 hours, culture media was completelychanged and flasks were incubated for 72 hours. Media was collected,centrifuged and stored at −80° C. until analysis for cytokines usingcommercially available colorimetric ELISA assay kits. For analysis ofsecreted cytokine release, sister flasks were treated with 10 mg/mLTNF-alpha, added during the last 24 hours of the 72 hour incubation. Foreach, lot three flasks of cells and supernatant were prepared, processedand banked independently for the basal and stimulated conditions,designated Basal Flask A, B and C or Stimulated Flask A, B and C,respectively.

Results show that when sub-cultured, ABM-SC secrete potentiallytherapeutic concentrations of several growth factors and cytokines knownto augment angiogenesis, inflammation and wound healing. See, FIG. 11.Hence, ABM-SC have been shown to consistently secrete several cytokinesand growth factors in vitro; including proangiogenic factors (e.g.,SDF-1 alpha, VEGF, ENA-78 and angiogenin), immunomodulators (e.g., IL-6and IL-8) and scar inhibitors/wound healing modulators (e.g., MMP-1,MMP-2, MMP-13 and Activin-A). Furthermore, the release of several ofthese factors is modulated by tumor necrosis factor alpha (TNF-alpha), aknown inflammatory cytokine released during the course of acute tissueinjury.

Example 3B Solid-Phase Capture and Identification of Secreted Factors(Table 1A, 1B and 1C)

Conditioned media were screened for the presence of various proteinssuch as cytokines, proteases, and soluble receptors by solid phaseantibody capture protein array, using RAYBIO™ Human Cytokine AntibodyArray (RayBiotech, Inc., Norcross, Ga., USA). Briefly, frozen aliquotsof conditioned media were thawed and warmed to room temperature prior touse. Array membranes were placed into the well of an eight-well tray (Cseries 1000). To each well, 2 mL 1× Blocking Buffer (RayBiotech, Inc.)was added and then incubated at room temperature for 30 min to block themembranes. Blocking Buffer was then decanted from each container, andthe membranes were then incubated with conditioned media (diluted 1:10with Blocking Buffer) at room temperature for 1 hr. Fresh cell culturemedia were used in place of PBS as negative controls. Samples were thendecanted from each container and washed 3 times with 2 mL of 1× WashBuffer I (RayBiotech, Inc.) at room temperature, while shaking for 5min. Array membranes were then placed into one well, with 1 mLbiotin-conjugated secondary antibody prepared in 1× Blocking Buffer, andincubated at room temperature for 1 hr. Arrays were then washed severaltimes with Wash Buffer. 2 mL HRP-conjugated streptavidin diluted 1:1000with 1× Blocking Buffer was added to each membrane and then incubated atroom temperature for 2 hrs. Membranes were then washed several timeswith 1× Wash Buffer. Detection reagents for chemiluminescence wereprepared as per manufacturer's instructions (RayBiotech, Inc.) andapplied to each membrane and incubated at room temperature for 2 minute.Membranes were then placed protein side up on a plastic sheet. Theopposite of the membrane was then covered with another piece of plasticsheet. Air bubbles were purged from the membranes by smoothing out theplastic. The membranes were then expose to x-ray film (Kodak X-OMAT AR™film) and then processed using a film developer.

Table 1A, 1B and 1C shows an extensive list of cytokines, growthfactors, soluble receptors, and matrix proteases secreted by humanABM-SC when sub-cultured in serum-free cell culture media. MediaSupernatant Concentrate #1=Advanced DMEM (Gibco™) supplemented with 4 mML-glutamine. Media Supernatant Concentrate #2=RPMI-1640 containing 4 mML-glutamine and HEPES (HyClone) supplemented withInsulin-Transferrin-Selenium-A (Gibco™)

The results demonstrate that numerous trophic factors and solublereceptors important for tissue regeneration and modulation of the immunesystem are produced by ABM-SC when cultured under these conditions.Notably, earlier experiments demonstrated that supplementation of thebase culture medium with insulin, transferrin, and selenium was requiredto achieve secreted protein levels such as those indicated in Table 1A,1B and 1C. Protein levels shown in Table 1A, 1B and 1C were assessedusing a RAYBIO™ Human Cytokine Antibody Array (RayBiotech, Inc.). Valuesare expressed as mean optical densities (O.D.). (N=2 for test samples.N=4 for controls.) Values reported with a (+) indicate mean O.D. valuesfor that particular analyte greater than two standard deviations abovethe mean O.D. values for the respective negative control. Valuesreported with a (−) represent mean O.D. values for that particularanalyte that are not greater than two standard deviations above the meanO.D. values for the respective negative control.

TABLE 1A Media Supernatent Media Supernatent Cytokine Concentrate #1Concentrate #2 POSITIVE CTL 11,020 (Mean O.D.) 11,127 (Mean O.D.) NEGCTL (Background) 2,360.00 2,271.00 Angiogenin 5800.5 (+) 4651 (+) BDNF5855.5 (+) 3587 (+) BLC 3852 (+) 3164.5 (+) BMP-4 3299 (+) 2610 (+)BMP-6 2359.5 (−) 2290.5 (−) CK beta 8-1 2408.5 (−) 2426 (−) CNTF 2655.5(+) 2663 (+) EGF 3932.5 (+) 2517 (+) Eotaxin 2527 (+) 2488 (+) Eotaxin-22467 (−) 2452.5 (+) Eotaxin-3 4564 (+) 4450 (+) FGF-6 2863.5 (+) 2883.5(+) FGF-7 2328 (−) 2374.5 (−) Flt-3 Ligand 2661 (+) 2414.5 (−)Fractalkine 2432.5 (−) 2379.5 (−) GCP-2 2546.5 (+) 2270 (−) GDNF 2299.5(−) 2208.5 (−) GM-CSF 2294 (−) 2129 (−) I-309 2431.5 (−) 2222 (−)IFN-gamma 2807.5 (+) 2848.5 (+) IGFBP-1 3192 (+) 4528.5 (+) IGFBP-24813.5 (+) 4244 (+) IGFBP-4 4640 (+) 4222.5 (+) IGF-I 2206.5 (−) 2238(−) IL-10 2225.5 (−) 2200.5 (−) IL-13 2582 (+) 2473 (+) IL-15 2472.5 (−)2622.5 (+) IL-16 2339.5 (−) 2229.5 (−) IL-1alpha 2698.5 (+) 2571.5 (+)IL-1beta 2276 (−) 2253 (−) IL-1ra 2609 (+) 2505.5 (+) IL-2 2523.5 (+)2381 (−) IL-3 2346 (−) 2270 (−) IL-4 2591 (+) 2402 (+) IL-5 3159 (+)3808 (+) IL-6 45570 (+) 40260.5 (+) IL-7 7336.5 (+) 5805 (+) Leptin 4187(+) 3733.5 (+) LIGHT 3689.5 (+) 3378.5 (+) MCP-1 9925.5 (+) 5561 (+)MCP-2 3117.5 (+) 2481.5 (+) MCP-3 2532 (+) 2382 (−) MCP-4 2702.5 (+)2694 (+) M-CSF 2387 (−) 2381.5 (−) MDC 2414.5 (−) 2510.5 (+) MIG 2344(−) 2342.5 (−) MIP-1-delta 2324 (−) 2259.5 (−) MIP-3-alpha 2323.5 (−)2261.5 (−) NAP-2 2517.5 (+) 2467.5 (+) NT-3 2973.5 (+) 3205.5 (+) PARC2668 (+) 2630 (+) PDGF-BB 2580.5 (+) 2780 (+) RANTES 2803 (+) 2760 (+)SCF 2765 (+) 2701.5 (+) SDF-1 3721 (+) 2562 (+) TARC 2488 (−) 2395 (−)TGF-beta 1 2381 (−) 2311 (−) TGF-beta 3 2422 (−) 2531 (+) TNF-alpha 2243(−) 2321 (−) TNF-beta 2355 (−) 2410.5 (−)

TABLE 1B Media Supernatent Media Supernatent Cytokine Concentrate #1Concentrate #2 POSITIVE CTL 12,318 (Mean O.D.) 11,936 (Mean O.D.) NEGCTL 2,452.00 2,392.00 Acrp30 2539.5 (+) 2436.5 (−) AgRP 2670 (+) 2494(−) Angiopoietin-2 3372 (+) 2656.5 (+) Amphiregulin 2692 (+) 2447 (−)axl 3398.5 (+) 3438.5 (+) bFGF 2915 (+) 2901.5 (+) Beta-NGF 2573.5 (+)2544 (+) BTC 2653.5 (+) 2554.5 (+) CCL28 2706.5 (+) 2553.5 (+) CTACK3502 (+) 3217 (+) dtk 2610.5 (+) 2512 (+) EGF-R 3057.5 (+) 2767.5 (+)ENA-78 2630.5 (+) 2503 (+) Fas/TNFRSF6 3312 (+) 3322.5 (+) FGF-4 2711(+) 2650.5 (+) FGF-9 2770 (+) 2538.5 (+) G-CSF 3950.5 (+) 3951 (+) GITRligand 2973.5 (+) 3107.5 (+) GITR 3198 (+) 2935 (+) GRO 29446.5 (+)10214 (+) GRO-alpha 7351 (+) 3553.5 (+) HCC-4 3241 (+) 2720.5 (+) HGF5535 (+) 3936.5 (+) ICAM-1 3043 (+) 2701.5 (+) ICAM-3 2621.5 (+) 2427(−) IGF-BP-3 3392 (+) 3190.5 (+) IGF-BP-6 5858 (+) 6111 (+) IGF-I SR2737.5 (+) 2757 (+) IL-1 R4/ST2 3463.5 (+) 3235.5 (+) IL-1 RI 2522.5 (+)2401 (−) IL11 2444.5 (−) 2273 (−) IL12-p40 2584 (+) 2536 (+) IL12-p702612 (+) 2618 (+) IL17 2610.5 (+) 2555.5 (+) IL-2 Ra 2491 (−) 2441.5 (−)IL-6 R 3202 (+) 2836 (+) IL8 24199.5 (+) 17594.5 (+) I-TAC 3898 (+) 3564(+) Lymphotactin 3415.5 (+) 3166 (+) MIF 3743 (+) 3524 (+) MIP-1-alpha2792 (+) 2747.5 (+) MIP-1-beta 2638.5 (+) 2523 (+) MIP-3-beta 2495.5 (−)2377 (+) MSP-a 2524.5 (+) 2394 (−) NT-4 2735 (+) 2635 (+)Osteoprotegerin 4183.5 (+) 3399 (+) Oncostatin M 2610 (+) 2508 (−) PlGF2705 (+) 2493 (−) sgp130 3232 (+) 2866.5 (+) sTNF RII 3124 (+) 3127 (+)sTNF-RI 9981 (+) 7929.5 (+) TECK 2887.5 (+) 2851 (+) TIMP-1 8718 (+)9342.5 (+) TIMP-2 11927 (+) 12602 (+) TPO 3712 (+) 3141.5 (+) TRAIL-R33129 (+) 3051 (+) TRAIL-R4 3417 (+) 3381 (+) uPAR 9557.5 (+) 8158.5 (+)VEGF 8587.5 (+) 6851 (+) VEGF-D 3477 (+) 3190.5 (+)

TABLE 1C Media Supernatent Media Supernatent Cytokine Concentrate #1Concentrate #2 POS 16,092 (Mean O.D.) 15,396 (Mean O.D.) NEG 2,338 1,747Avtivin A 23239.5 (+) 18339 (+) ALCAM 14185.5 (+) 15463.5 (+) B7-1(CD80) 2983.5 (+) 2222.5 (+) BMP-5 2770.5 (+) 2011.5 (+) BMP-7 2564 (+)1828 (−) Cardiotrophin-1 2816.5 (+) 2097 (+) CD14 3556 (+) 2334.5 (+)CXCL-16 4108.5 (+) 2559 (+) DR6 (TNFRSF21) 3477 (+) 2312 (+) Endoglin3070 (+) 2135 (+) ErbB3 3366 (+) 2313.5 (+) E-Selectin 2846.5 (+) 1918(+) Fas-Ligand 3531.5 (+) 2943.5 (+) ICAM-2 3158.5 (+) 2155.5 (+) IGF-II3212 (+) 2395.5 (+) IL-1 R II 2855 (+) 1834 (−) IL-10 Rb 2780 (+) 1916(+) IL-13 Ra2 2559.5 (+) 1693 (−) IL-18 BPa 2921 (+) 1881 (−) IL-18 Rb3238.5 (+) 2387 (+) IL-2 Ra 3666 (+) 2316.5 (+) IL-2 Rb 3001 (+) 2083.5(+) IL-2 Rg 3121 (+) 2185.5 (+) IL-21R 3567.5 (+) 2534.5 (+) IL-5 Ra3084.5 (+) 2237 (+) IL-9 3676 (+) 2324.5 (+) IP-10 3300.5 (+) 2262.5 (+)LAP 6202 (+) 5383.5 (+) Leptin R 3487 (+) 2791 (+) LIF 3486.5 (+) 2400.5(+) L-Selectin 3036.5 (+) 2160 (+) M-CSF R 3140 (+) 2330.5 (+) MMP-13469 (+) 2499 (+) MMP-13 3083.5 (+) 2316.5 (+) MMP-9 3058.5 (+) 2370 (+)MPIF-1 2974 (+) 2274.5 (+) NGF R 2887.5 (+) 2355 (+) PDGF-AA 4130 (+)3423.5 (+) PDGF-AB 3191.5 (+) 2278.5 (+) PDGF Ra 4430 (+) 4027 (+) PDGFRb 3768 (+) 2784 (+) PECAM-1 4071.5 (+) 3450 (+) Prolactin 3199.5 (+)2151 (+) SCF R 3431.5 (+) 2668.5 (+) SDF-1b 2268.5 (−) 2156 (+) Siglec-52691 (+) 2160.5 (+) TGF-a 3058.5 (+) 2388.5 (+) TGF b2 3316 (+) 2583 (+)Tie-1 2883 (+) 3178 (+) Tie-2 3565 (+) 3802.5 (+) TIMP-4 6468 (+) 6248(+) VE-Cadherin 3164.5 (+) 2428 (+) VEGF R2 4030.5 (+) 3003 (+) VEGF R33200 (+) 2651.5 (+)

Example 4 Bioactivity of Adult Bone Marrow-Derived Somatic Cells: InVitro Neurogenesis Enhanced by Secreted Factors

A stock solution of collagen was first prepared by re-suspending rattail collagen (Sigma Chemical) in 0.1N acetic acid at a finalconcentration of 3.0 mg/mL. The collagen-based medium then was preparedas described by Bell et al., Proc. Natl. Acad. Sci. USA, vol. 76, no. 3,pp. 1274-1278 (March 1979) with minor modifications as described herein.Briefly, the collagen medium was prepared by mixing the rat tailcollagen solution with DMEM 5× (JRH Biosciences) supplemented with 5 mML-glutamine (CELLGRO™), Antibiotic-Antimycotic Solution (CELLGRO™), anda buffer solution (0.05N NaOH (Sigma Chemical), 2.2% NaHCO₃ (SigmaChemical), and 60 mM HEPES (JRH Biosciences) at a ratio of 4.7:2.0:3.3.Approximately 500 microL of the collagen cell suspension was added toeach well of a 24-well culture plate. The 24-well plates were thenplaced in a 37° C. humidified trigas incubator (4%0₂, 5% CO₂, balancedwith nitrogen) for 1 hour to permit the collagen solution to congeal.Frozen rat PC-12 were thawed, washed in RPMI-1640 supplemented with 4 mML-glutamine and HEPES (HYCLONE™) supplemented withInsulin-Transferrin-Selenium-A (GIBCO™) and centrifuged at 350×g for 5minutes at 25° C. Cell pellets were re-suspended in same solution at aconcentration of 75,000 viable cells/mL, with and without 136 ng/mL ratbeta-NGF (β-NGF) (Sigma Chemical), 1:50 dilution of unconditionedconcentrated RPMI-1640/ITS medium (used as a negative control), and a1:50 dilution of conditioned concentratedRPMI-1640/Insulin-Transferrin-Selenium-A (ITS) media (media wasconditioned as described in Example 1; conditioned and unconditioned,negative control media were concentrated as described in Example 1).Next, 1 mL of cell suspension was dispensed evenly across the surface ofeach of 2 gels (1 mL gel) for each cohort and then verified by phasecontrast microscopy. The plates were then placed in a 37° C. humidifiedtrigas incubator (4% O₂, 5% CO₂, balanced with nitrogen). Spent culturemedia was replaced every 3 days with fresh media. Images were capturedon Day 10. See, FIG. 2.

These results demonstrate that PC12 differentiation into neurons by NGFis augmented dramatically when supplemented with conditioned mediaproduced by human ABM-SC. Interestingly, the extent of neuraldifferentiation, as assessed by the number of axon and neurites in theculture, was not significant when conditioned media was added alone.While some neurite outgrowth was observed in the presence of NGF alone,supplementing the cultures with conditioned media dramatically increasedboth the number and length of neurites. Previous work in our lab showedthat supplementing RPMI culture media with insulin, transferrin, andselenium was critical for neural differentiation of PC12 under allstandard published experimental conditions tested. These data indicatethat media conditioned by human ABM-SC contain components whichsupplement or induce neurite outgrowth over and above the levelsobtained with RPMI/ITS media alone or with RPMI/ITS media containingNGF. See, FIG. 2.

Example 5 Bioactivity of Adult Bone Marrow-Derived Somatic Cells:Inhibition of Mitogen-Induced T Cell Proliferation In Vitro

Human ABM-SC (Lot #RECB801 at ˜18 population doublings) and exABM-SC(RECB906 at ˜43 population doublings), were plated in 75 cm² flasks at aconcentration of 6000 viable cells/cm² in complete media (MinimalEssential Medium-Alpha (HYCLONE™) supplemented with 4 mM glutamine and10% sera-lot selected, gamma-irradiated, fetal bovine serum (HYCLONE™)and incubated at 37° C. in a humidified trigas incubator (4% O₂, 5% CO₂,balanced with nitrogen). After 24 hrs, spent media was aspirated andreplaced with 15 mL fresh media. Human mesenchymal stem cells (hMSC,Catalog #PT2501, Lot #6F3837; obtained from Cambrex ResearchBioproducts; now owned by Lonza Group Ltd., Basel, Switzerland) wereplated in 75 cm² flasks at a concentration of 6000 viable cells/cm² in15 mL Mesenchymal Stem Cell Growth Medium (MSCGM™; Lonza Group Ltd.,Basel, Switzerland) and incubated at 37° C. in a humidified incubator atatmospheric O₂ and 5% CO₂. After 24 hrs, spent media was aspirated andreplaced with 15 mL fresh MSCGM™. Both human ABM-SC (hABM-SC) and hMSCwere harvested after 96 hours in culture. Harvested hABM-SC and hMSCwere plated in 96-well round bottom plates at a concentration of 25,000viable cells/mL in RPMI-complete media (HYCLONE™) Human peripheral bloodmononuclear cells (PBMCs) were labeled in 1.25 microM CarboxyFluorosceinSuccinimidyl Ester (CFSE) and cultured at 250,000 cells/well inRPMI-complete media along with hMSC, Lot #RECB801, Lot #RECB906 hABM-SCor alone. To stimulate T cell proliferation, cultures were inoculatedwith 2.5 or 10 microg/mL Phytohaemagglutinin (Sigma Chemical). Cellswere then harvested 72 hrs later and stained with CD3-PC7 antibody(Beckman Coulter), as per manufacturer's instructions, and analyzed on aBeckman FC 500 Cytometer, using FlowJo 8.0 software (Tree Star, Inc.,Ashland, Oreg.). Only CD3+ cells were analyzed for division index. See,FIG. 3.

These findings demonstrate that exABM-SC possess the capacity to inhibitT cell activation and proliferation and, therefore, may be useful as atherapeutic to suppress T cell-mediated graft rejection, autoimmunedisorders involving dysregulation of T cells, or to induce a state ofimmune tolerance to an otherwise immunogenic skin product. Thus, onecould envision the use of allogeneic human exABM-SC or compositionsproduced by such cells, to treat burn patients awaiting surgicalapplication of an allogeneic skin product. In such an embodiment,treating an open wound first with exABM-SC, or compositions produced bysuch cells, may act not only to help rebuild the wound bed by incitinghost cells to migrate to the cite of injury, but also to provide anenvironment permissive to long term engraftment of allogeneic skin orskin substitutes.

Example 6 Reconstitution of Porcine ABM-SC in Aqueous Vehicle for InVivo Administration

Porcine ABM-SC were seeded at 60 cells/cm², refed at day 4, and grownfor a total of 6 days. Cells were collected and frozen until subsequentuse. Frozen aliquots of porcine ABM-SC were thawed, washed in DPBSG(Dulbecco's Phosphate Buffered Saline (CELLGRO™)) supplemented with 4.5%glucose) and centrifuged at 350×g for 5 minutes at 25° C. Cell pelletswere re-suspended in DPBSG at a concentration of approximately50,000/microL. Cell counts and viability assays were performed using aCOULTER™ AcT 10 Series Analyzer (Beckman Coulter, Fullerton, Calif.) andby trypan blue exclusion, respectively. The cell suspension was thenloaded into a 1cc tuberculin syringe.

Example 7 Bioactivity of Adult Bone Marrow-Derived Somatic Cells:Treatment of Incisional Wounds with Allogeneic Porcine ABM-SC

Two Yucatan swine, weighing between 57 kg and 78 kg were anesthetizedand prepared for aseptic surgery. Four incisional wounds measuringapproximately 50 mm in length were made with a scalpel blade on bothsides of two animals (Nos. 3 and 4) for a total of eight wounds peranimal along the paravertebral and thoracic area skin. Bleeding wasstopped by inserting sterile gauze soaked with epinephrine into thelesion site. Gauze was then removed after about 10-20 minutes and eachwound was treated with a single dose of porcine ABM-SC, divided into 12separate injections evenly spaced around the incision with an additional10-300 microL applied to the wound bed itself Control wounds wereinjected similarly with vehicle only (DPBSG). Wounds were then closedwith Steri-Strips™ (3M) and the animals were covered with protectivealuminum jackets. The jackets were checked several times each day toensure stable and proper position. The wound dressings were monitoreddaily and changes photographed on days 0, 1, 3, 5, and 7. Animals wereeuthanized on day 7 for histopathology. Formalin fixed paraffin embeddedtissue sections were prepared and stained by H&E. Histomorphometricscoring was conducted by an expert veterinary pathologist blinded to thetreatment group.

Seven days following treatment of the wounds, lesions treated withallogeneic porcine ABM-SC shown almost no signs of visible scarring(FIG. 4) while those treated with vehicle exhibited visible signs ofscarring. Histomorphometric analysis of the wounds showed a markedreduction in tissue macrophages (histiocytes) in those treated with theABM-SC, while no significant difference was seen in any of the otherhistological scores assessed.

When similar tissue sections were scored for the extent ofre-epithelialization (a crude indicator wound healing rate), thosetreated with ABM-SC exhibited a marked increase in the amount ofepithelial cells repopulating the site of the incisions (FIG. 5).

Example 8 Bioactivity of Human ABM-SC in Collagen Vehicle for In VivoAdministration as a Liquid, Semi-Solid, or Solid-Like Therapeutic (FIG.6-9)

When reconstituted in a collagen-based biodegradable vehicle and storedat 4° C., human ABM-SC (Lot # PCH610; ˜27 population doublings) retainhigh cell viability for at least 24 hours (as demonstrated by cellbioactivity in gel contraction assays). Stored this way, the collagensolution will remain as a liquid and will preserve the cells in asuspended state without significant loss of viability (FIG. 6).Bioactivity of the cells can then be assessed using an in vivo assay ofwound repair. To conduct this assay, a stock solution of collagen wasfirst prepared by re-suspending rat tail collagen (Sigma Chemical) in0.1N acetic acid at a final concentration of 3.0 mg/mL. The collagenmedium was prepared as described by Bell et al. (Proc. Natl. Acad. Sci.USA, vol. 76, no. 3, pp. 1274-1278 (March 1979)) with minormodifications as described herein. Briefly, the collagen medium wasprepared by mixing the rat tail collagen solution with DMEM 5× (JRHBiosciences) supplemented with 5 mM L-glutamine (CELLGRO™),Antibiotic-Antimycotic Solution (CELLGRO™; Catalog #30-004-C1), and abuffer solution (0.05N NaOH (Sigma Chemical), 2.2% NaHCO₃ (SigmaChemical), and 60 mM HEPES (JRH Biosciences) at a ratio of 4.7:2.0:3.3.Frozen human adult bone marrow derived somatic cells (hABM-SC) werethawed, washed in DMEM 1× and centrifuged at 350×g for 5 minutes at 25°C. The cell pellets were re-suspended in DMEM 1× at concentration ofapproximately 72,000 total cells/microL. Fifty microliters of cellsuspension was then added to 2 mL collagen medium and gently triturated(i.e., gently pipetted up and down to obtain a homogeneous suspension ofcells in collagen medium), yielding a final cell concentration ofapproximately 1,800 cells/microL. The cell suspension was then stored atapproximately 4-8° C. overnight. The following day, the liquid cellsuspension was transferred from the 15 mL conical tube and dispensedinto 24-well cell culture plates at approximately 500 microL/well. Theplates were then placed in a 37° C. humidified trigas incubator (4% O₂,5% CO₂, balanced with nitrogen) for 1 hour to permit the collagen tosolidify into a semi-solid gel. The gels were then removed from the24-well plates using disposable sterile spatulas (VWR) and transferredto 12-well culture plates. The gels were then floated in 1.0 mL DMEM 1×per well. For negative controls, gels were prepared as described butwithout cells. Three wells were seeded for each condition (n=3).

To evaluate the extent to which gel contraction is dose-dependent, asimilar assay was conducted wherein human exABM-SC (Lot# RECB819; at ˜43population doublings) were reconstituted in collagen solution atdifferent cell concentrations immediately after removal from cryostorage(FIG. 7). A stock solution of collagen was first prepared byre-suspending rat tail collagen (Sigma Chemical) in 0.1N acetic acid ata final concentration of 3.0 mg/mL. The collagen medium then wasprepared as described by Bell et al. (1979) with minor modifications asdescribed herein. Briefly, the collagen medium was prepared by mixingthe rat tail collagen solution with DMEM 5× (JRH Biosciences)supplemented with 5 mM L-glutamine (CELLGRO™) Antibiotic-AntimycoticSolution (Cellgro™), and a buffer solution (0.05N NaOH (Sigma Chemical),2.2% NaHCO₃ (Sigma Chemical), and 60 mM HEPES (JRH Biosciences)) at aratio of 4.7:2.0:3.3. Frozen human adult bone marrow derived somaticcells (hABM-SC) were thawed, washed in DMEM 1× and centrifuged at 350×gfor 5 minutes at 25° C. The cell pellets were re-suspended in DMEM 1× atconcentration of approximately 40,000, 80,000 and 200,000 viablecells/microL. Fifty microliters of each cell suspension was added to 2mL collagen medium and gently triturated. Approximately 500 microL ofthe collagen cell suspension was added to each well of a 24-well cultureplate. The plates were then placed in a humidified 37° C. trigasincubator (4% O₂, 5% CO₂ balanced with nitrogen) for 1 hour to permitthe collagen solution to solidify. The gels were then removed from theplates using disposable sterile spatulas (VWR) and transferred to12-well culture plates. The gels were floated in 1.0 mL DMEM 1× perwell.

As a negative control, gels were prepared as described above using thehighest concentration of hABM-SC (5×10⁶/mL) except that the cells wereheat-inactivated (to eliminate biological activity). Heat-inactivatedcells were first prepared by heating the initial cell suspension in DMEM1× medium to 70° C. in a heat block containing water (heat transfer) for40 minutes. Three wells were seeded for each condition (n=3).

To determine the extent to which the gels contracted over time, thepercentage initial or starting surface area was calculated from digitalimages captured at 0, 24, 48 and 72 hours using a flatbed scanner. Fromeach image, the diameter of the gel was measure both horizontally andvertically and then averaged. Results demonstrate that both the rate andextent of gel contraction was effected in a dose dependent manner (FIG.7).

To determine the levels of certain secreted proteins produced from thehuman ABM-SC in these semi-solid gels, enzyme-linked immunosorbant assay(ELISA) was performed (on day 3 of culture) on conditioned cell culturesupernatants collected from the liquid media surrounding the gels (FIG.8). Supernatants were transferred to sterile 15 mL conical tubes andcentrifuged at 1140×g for 15 minutes to remove cell debris. Supernatantswere then transferred to 2 mL cryovials and transferred to −80° C. forshort-term storage. On the day of assay, supernatants were thawed andequilibrated to room temperature before use. ELISA analysis wasperformed to detect IL-6, VEGF, Activin-A, pro-MMP-1, and MMP-2 ELISA(conducted as per manufacturer's instructions; all kits were purchasedfrom R&D Systems, Inc. (Minneapolis, Minn., USA)). Results demonstratethat therapeutically relevant levels of trophic factors can be producedby these semi-solid neotissues and that these levels can be controlledby adjusting cell concentration. Of the trophic factors measured,detectable levels were not seen in cultures containing heat inactivatedcells only. Statistical comparisons between assay conditions weredetermined by One-way ANOVA (*** p<0.001).

Human ABM-SC can also be reconstituted in a collagen solution toconstruct a large-format semi-solid structure that could be used astopical therapeutic (FIG. 9). To construct such a structure, a stocksolution of collagen was first prepared by re-suspending rat tailcollagen (Sigma Chemical) in 0.1N acetic acid at a final concentrationof 3.0 mg/mL. The aqueous collagen medium was prepared by mixing the rattail collagen solution with DMEM 5× (JRH Biosciences) supplemented with5 mM L-glutamine (CELLGRO™), Antibiotic-Antimycotic Solution (CELLGRO™)and a buffer solution (0.286N NaOH (Sigma Chemical), 1.1% NaHCO₃ (SigmaChemical), and 100 mM HEPES (JRH Biosciences) at a ratio of 6:2:2.Frozen hABM-SC were thawed and washed in 1×DMEM and then centrifuged at350×g for 5 minutes at 25° C. The cell pellet was re-suspended in 1×DMEM at a concentration of approximately 90,000 cells/microL.Approximately 1.1 mL of cell suspension was then added to 20 mL collagenmedium and gently triturated to achieve a final cell concentration of5×10⁶ cells/mL. The final concentration of collagen was 1.8 mg/mL. Thecell suspension was then dispensed into a 10 cm Petri dish (formingdish). The effective dose of cells in the collagen solution dispensedwas approximately 100×10⁶ viable cells. The 10 cm forming dishcontaining the cell suspension was then placed in a humidified 37° C.incubator (5% CO₂) for 1 hour to permit the collagen solution tosolidify. The semi-solid gel was then carefully removed from the 10 cmforming dish and transferred to a 15 cm Petri dish (culture dish) andphotographed.

To construct a solid-like neotissue derived from human ABM-SC andcollagen, the semi-solid structure described above can be placed backinto a 37° C. humidified cell culture incubator (5% CO₂) for anadditional 2 days (FIG. 10). To form a solid-like neotissue, asemi-solid gel prepared as described above, with the exception that thefinal collagen solution was 1.4 mg/mL (instead of 1.8 mg/mL), wascarefully dislodged from the edges of the 10 cm forming dish and floatedin approximately 82 mL 1×DMEM containing Antibiotic-Antimycotic Solution(CELLGRO™) in a 15 cm culture dish. The semi-solid gel was thentransferred to a 37° C. humidified incubator (5% CO₂) for an additional48 hrs to facilitate remodeling of the matrix into a solid-like tissuestructure, free of the starting collagen substrate. The solid-likeneotissue was then removed from the 15 cm culture dish and photographed(FIGS. 10A and 10B). Histological analysis of the neotissue by Masson'sTrichrome stain demonstrates that the matrix is rich in newly synthesizehuman collagens and proteoglycans (FIG. 10C). Control collagen gels donot stain by this method. Collagens and proteoglycans stain blue.

The results of these studies indicate that frozen stocks of ABM-SC canbe dispensed upon thaw and reconstituted in a liquid collagen-basedmedium that could be used therapeutically as a liquid suspension,semi-solid construct, or solid-like neotissue. When prepared in such away and stored at approximately 4-10° C., the cell suspension willremain as liquid while maintaining satisfactory cell viability forgreater than 24 hours. Employing such a method to formulate ABM-SC forclinical application then would provide considerable latitude to theclinician administering the cells. The suspension could be administeredas a liquid injectable or, alternatively, could be applied topically toa wound bed. In the latter case, the liquid cell suspension would beanticipated to mold to the contour of the wound and then congeal into asemi-solid structure (for example, when warmed to ˜37 degrees C.).Alternatively, the suspension could be used in such a way as tomanufacture semi-solid constructs or solid-like neotissues.

These data also show that when prepared by the methods, the resultingcompositions each possess bioactivity important for mediating repair ofvarious types of wounds, particularly those involving the skin.

ExCF-SC (for example, exABM-SC), or compositions produced by such cells,prepared in a liquid collagen-based medium could therefore be usedtopically to treat open wounds or as an injectable alternative to dermalfillers for facial rejuvenation.

In the semi-solid form, exCF-SC (for example, exABM-SC) or compositionsproduced by such cells, cold be used topically to treat severe burnpatients that have had damaged full-thickness skin removed surgically,thereby acting as a dermal replacement.

Solid neotissues produced by exCF-SC (for example, exABM-SC) could beused surgically as an alternative to human cadaveric skin (ALLODERM™),porcine skin (PERMACOL™) and other animal-derived constructs (INTEGRA™).Moreover, these data also show that the potency of each of these variousconstructs can be controlled by altering dose of cells or compositionsproduced by the cells.

Example 9 Improvement of Cardiac Function in Rats Treated with hABM-SC

Administration of human ABM-SC to animals following myocardial infarctdemonstrates that CF-SC (such as ABM-SC) improve cardiac function andenhance repair of cardiac tissue damage by stimulating angiogenesis andreducing fibrosis. See, FIG. 15. A rat model for acute myocardialinfarction was utilized by occluding a coronary artery thereby creatinga cardiac lesion (i.e., damaged region of heart). Lesioned rats wereinjected intercardially with either hABM-SC or vehicle.

Heart Function Methods:

Sprague-Dawley rats of both sexes (age approx. 3 months) receivedexperimentally-induced myocardial infarction via the placement of apermanent silk ligature around the left-anterior descending (LAD)coronary artery via a midline sternotomy. Five days after thisprocedure, the rats were begun on a standard regimen of Cyclosporine Atreatment that lasted for the duration of the study. On day 7-8following infarction, rats were anesthetized, intubated and anintercostal incision was made to expose the apex of the heart. Anultrasonic Millar catheter was then inserted through the ventricularwall, and pressure over time measurements were recorded for a period ofapproximately 30-60 seconds. This model of infarct production andpressure/time measurements of cardiac function is a standard, wellcharacterized model by which the effects of cellular therapies oncardiac function can be assessed (See e.g., Müller-Ehmsen, et al.,Circulation., 105(14):1720-6 (2002)).

The test composition was delivered using a 100 microL Hamilton syringefitted with a 30 gauge, low dead-space needle. Five separate injectionsof 20 microL were performed over the course of 2-3 minutes. Fourinjections were performed at equal distances around the visualizedinfarct, while the fifth was placed directly into the center of theinfarcted region as determined by area of discoloration. Afterinjection, the incision was sutured closed, the pneumothorax wasreduced, and the animals were weaned from the respirator and extubated.Four weeks after injection (5 weeks post-infarction), animals werereanesthetized, the heart was exposed through a midline sternotomy, anda Millar catheter was inserted. Dp/dt measurements were taken asdescribed above, after which the rats were euthanized viaexsanguination.

Heart Function Results (FIG. 13):

Four weeks after treatment, rats receiving ABM-SC demonstratedsignificantly higher +dp/dt (peak positive rate of pressure change)values (A). Expressing changes in cardiac function over the course ofthe study by subtracting 0 week +dp/dt values from 4 week values (“delta+dp/dt”) demonstrated that while vehicle treated rats had decreases incardiac function over the course of the study (negative delta), animalstreated with either cell preparation showed significant improvement incardiac function (B). Compared to vehicle treated rats, those receivingABM-SC demonstrated significantly lower tau values (C), suggestingincreased left ventricular compliance. Tau is the time constant ofisovolumetric left ventricular pressure decay. For peak negative rate ofpressure change (−dp/dt), expressing changes in cardiac function overthe course of the study by subtracting 0 week −dp/dt values from 4 weekvalues (“delta −dp/dt”) demonstrated that while vehicle-treated rats haddecreases in cardiac function over the course of the study (negativedelta), animals treated with cell preparation showed significantimprovement in cardiac function (D). [*p<0.05, **p<0.01 by ANOVA]

Heart Structure Methods:

Sprague-Dawley rats received experimentally-induced myocardialinfarction via the placement of a permanent silk ligature around theleft-anterior descending (LAD) coronary artery. Animals received astandard regimen of Cyclosporine A treatment (10 mg/kg s.c. daily) thatlasted for the duration of the study.

On day 7-8 following infarction, rats were anesthetized, intubated andan intercostal incision was made to expose the apex of the heart.Cardiac function was accesses after which the test article was deliveredusing a 100 microL Hamilton syringe fitted with a 30 gauge, lowdead-space needle. Five separate injections of 20 microL were performedover the course of 2-3 minutes. Four injections were performed at equaldistances around the visualized infarct, while the fifth was placeddirectly into the center of the infracted region as determined by areaof discoloration. After injection, the incision was sutured shut, thepneumothorax was reduced, and the animals were weaned from therespirator and extubated. Four weeks after injection (5 weekspost-infarction), animals were reanesthetized, the heart was exposedthrough a midline sternotomy, and cardiac function accessed. Afterfunctional measures were completed rats were euthanized viaexsanguination. Rats were first deeply anesthetized using a mixture ofketamine (75 mg/kg) and medetomidine (0.5 mg/kg). The thoracic cavitywas then surgically exposed and the heart dissected and immersion fixedin 10% neutral buffered formalin. Hearts were then grossly sectionedinto three pieces, oriented into embedding molds, and processed forparaffin embedding. Heart tissues were then sectioned at 6 μm andstained by Hemotoxylin & Eosin (H&E) or Masson's Trichrome. At least sixsections from every heart were also stained with hemotoxylin/eosin andTrichrome respectively. Specifically, trichrome staining allows for thevisualization of collagen (blue) versus muscle tissue (red). Sincecollagen indicates the presence of scar tissue (absence ofregeneration), the ratios of collagen to normal cardiac muscle weredetermined A semiquantitative scoring scale was devised, with 0 as nodetectable collagen and 5 as maximal/severe. Stained sections were thensent to a board certified pathologist for histomorphometric scoring.

Each slide contained three cross-sections of the heart, demonstrating across-sectional view of both ventricles from the mid-ventricular area(1) distal ⅓ of the ventricle (2), and apex of the ventricle (3). Forhistomorphometric analyses, the following grading scheme was used:

Location of Tissue Damage:

Left ventricle (LV), Right ventricle (LV), Both ventricles (BV).

Percent of affected ventricle damaged (size of injury): Given in percent(0-100%)

Thickness Score of Experimentally Damaged Area of Ventricle:

Given a grade of 1-4 based on estimated thickness in millimeters.Compared with known landmarks in the tissue sections (e.g. averageerythrocyte is 7 microns in diameter; average myocardial muscle bundleis 30 microns in diameter). Grade 1 (less than 5 mm); Grade 2 (0.5 mm to1 mm); Grade 3 (1 mm to 1.5 mm); Grade 4 (1.5 mm +).

Neovascularization in area of tissue damage: (Grade of 0 to 4, fromnormal (0) to neovascularization throughout the entire area of initialtissue damage (4).

Initial Vascular Damage:

Includes degeneration/necrosis of pre-existing blood vessels, withthrombosis and/or inflammation resulting from removal of remainingvascular debris, expressed as a grade of 0 to 4, with 0 being novascular damage present, and 4 being vascular damage throughout theaffected area.

Extent of Fibrosis within the Area of Tissue Damage:

Expressed as a grade of 0 to 4, from no fibrosis (0) to (4) in 20%graduating levels of fibrosis and scarring of the initial area of damagecaused by the infarction procedure. For example, fibrosis of 20% of theventricle would be assigned a grade of (1), and fibrosis of 40% of theventricle would be assigned a grade of (2), 60% would receive a (3), andabove 60% would receive a (4).

Heart Structure Results:

Rats were subsequently sacrificed and cardiac tissue was sectioned andstained. A board certified veterinary pathologist performedsemiquantitative scoring (FIG. 15) to evaluate changes in infarct sizein the hearts of rats receiving vehicle or ABMSC seven days aftermyocardial infarction. Histopathological analysis indicated significantreduction in infarct size in rats receiving hABM-SC compared to vehicle.According to a preset scale, rats receiving hABM-SC had histologicalscores approximately two points lower than vehicle controls. FIG. 14shows an example of typical infarct size reduction. Histopathologicalanalysis determined that hABM-SC reduced fibrosis and increasedvascularization in the infarct zone (FIG. 15), consistent withpro-regenerative activity. Thus, it was observed that rats treated withhABM-SC showed dramatic improvement of cardiac tissue structure. See,FIGS. 14 and 15.

Example 10 Adult Bone Marrow-Derived Somatic Cells Suppress ImmuneMediated Responses

Part I: Suppression of Mitogen-Induced T-Cell Proliferation in One-WayMLR (Mixed Lymphocyte Reaction) Assay.

Methods:

Human ABM-SC and exABM-SC (Lot #RECB801 and RECB906, respectively), wereplated in 75 cm² flasks at a concentration of 6000 viable cells/cm² in15 mL complete media such as Advanced DMEM (GIBCO™; Catalog #12491-015,Lot #1216032 (Invitrogen Corp., Carlsbad, Calif., USA)) supplementedwith 4 mM L-glutamine (Catalog #SH30034.01. Lot #134-7944, (HYCLONE™Laboratories Inc., Logan, Utah, USA)) or HyQ® RPMI-1640 (HYCLONE™Catalog #SH30255.01, Lot # ARC25868) containing 4 mM L-glutamine andsupplemented with Insulin-Transferrin-Selenium-A (ITS) (GIBCO™; Catalog#51300-044, Lot #1349264) and incubated at 37° C. in a humidified trigasincubator (4% O₂, 5% CO₂, balanced with Nitrogen). After 24 hrs, spentmedia was aspirated and replaced with 15 mL fresh media. Humanmesenchymal stem cells (hMSC) (Lonza BioScience, formerly CambrexBioscience, Catalog #PT2501, Lot #6F3837) were plated in 75 cm² flasksat a concentration of 6000 viable cells/cm² in 15 mL MSCGM™ (LonzaBioScience) and incubated at 37° C. in a humidified incubator atatmospheric O₂ and 5% CO₂. After 24 hrs, spent media was aspirated andreplaced with 15 mL fresh MSCGM™. Both hABM-SC and hMSC were harvestedafter 96 hours in culture. Harvested hABM-SC and hMSC were plated in96-well round bottom plates at a concentration of 25,000 viable cells/mLin RPMI-complete media (Hyclone). Human peripheral blood mononuclearcells (PBMCs) were labeled in 1.25 uM CarboxyFluoroscein SuccinimidylEster (CFSE) and cultured at 250,000 cells/well in RPMI-complete mediaalong with hMSC, RECB801, RECB906 or alone. To stimulate T cellproliferation, cultures were inoculated with 2.5 or 10 micrograms/mLPhytohaemagglutinin (Sigma Chemical). Cells were then harvested after 72hrs later and stained with CD3-PC7 antibody (Beckman Coulter), as permanufacturer's instructions, and analyzed on a Beckman FC 500 Cytometer,using Flow Jo software. Only CD3+ cells were gated analyzed for divisionindex.

Results:

Allogeneic human ABM-SC and exABM-SC suppress mitogen-induced T-cellproliferation in one-way MLR assay. See, FIG. 16.

Part II: Allogeneic Porcine ABM-SC Fail to Illicit T-Cell MediatedImmune Response In 2-Way MLR Challenge Assay.

Methods:

Porcine whole blood was collected for immunoassays on Day 0 (prior totreatment) and at necropsy (Day 3 or Day 30 post-treatment) for cellularimmune response analysis. PBMC from each animal were cultured withpABM-SC, the mitogen ConA, or media alone. Samples were analyzed by flowcytometry and the amount of proliferation calculated using FlowJosoftware.

Whole blood samples were diluted 1:1 with DPBS (Dulbecco's PBS)-2%Porcine Serum. Diluted blood was overlayed on Ficoll (2:1 ratio dilutedblood to Ficoll) and centrifuged at 350×G for 30 minutes, withcentrifugation cycle ending with zero braking. The resulting top layerwas aspirated. The middle layers, which contain the desired mononuclearcells, were pooled for each sample, and washed in 3× with DPBS-2%Porcine Serum. After washing, the pellet was resuspended in 20 mL ACKlysis buffer and incubated for 3 minutes, to remove residual red bloodcells, then centrifuged for 5 minutes, at 250×G. The pellets were washedin 20 mL DPBS-2% Porcine Serum and resuspended in 5 mL RPMI Completemedia (RPMI-1640, 10% Porcine serum, 2 mM L-glut, 20 mM HEPES, 0.1 mMNEAA, 1× Penn-Strep). Cells were frozen at a concentration of 20×10⁶cells/mL by centrifugation, and resuspension in ice cold freeze media(10% DMSO in Porcine Serum) and immediately added to 2 mL cryovials andplaced into a cryorate freezer. (freeze rate=−25° C./min to −40° C.,+15° C./min to −12.0° C., −1° C./min to −40° C., −10° C./min to −120°C.). Cells were stored in 2 mL aliquots per vial in vapor phase ofliquid nitrogen until use.

On day 0, pABM-SC were plated in 96 well culture dishes at aconcentration of 10,000 cells/well in AFG-104 media according to studytemplate for each test condition. Plates were incubated overnight at 37°C. in a humidified incubator with low O₂(4-5%), ˜5% CO₂ balanced withnitrogen.

The following day, PBMC were labeled with CFSE (carboxy-fluoresceindiacetate, succinimidyl ester). In short, thawed vials of PBMC in 37° C.water bath, washed with 10 mL RPMI-Complete media centrifuged cells at300×G, and resuspended in DPBS. Cell concentrations were adjusted to10×10⁶ cells/mL and incubated with CFSE at a final concentration of0.625 mM for 5 minutes. Cells were immediately washed in 40 mL ice coldDPBS/5% porcine serum and centrifuged 10 minutes at 300×G. Cells wereagain washed in 25 mL DPBS/5% porcine serum and centrifuged as before.Cells were washed a third and final time in 10 mL RPMI-complete media.Cell concentrations were adjusted to a final concentration of 5×10⁶cells/mL. Labeled PBMC were added to the assay plate according to studytemplate as follows: AFG-104 media was aspirated and replaced with 100microL RPMI-Complete media. 100 microL of RPMI-complete media was addedto non-stimulated wells, 100 microL media with 20 microg/mL ConA inRPMI-Complete media was added to stimulated wells, and 4.5%Glucose-RPMI-Complete media to vehicle cells. 500,000 labeled PBMC wereplated per well in 96 well plates according to study template. Plateswere incubated for 5 days at 37° C., atmospheric O₂ (high O₂), withhumidity, 5% CO₂ no nitrogen. All conditions were completed intriplicate for each blood sample received. Vehicle stimulation wascompleted for a subset of blood samples, but was not significantlydifferent than media alone. After 5 days of co-culture, cells wereharvested for flow cytometry by transferring cells from 96 well plate toa flow tube. Indirect staining was conducted in accordance with standardprotocols. The primary antibody used was Biotin Conjugated Mouseanti-Pig CD3 Monoclonal antibody; followed by exposure toStreptavidin-PE-Cy7 secondary reagent. Cells were resuspended in 200microL flow wash buffer and analyzed on a Coulter FC500 device.

Results:

A Division Index was calculated for samples collected at baseline and at3 or 30 days post-treatment and then challenged in vitro with media,vehicle, pABM-SC or ConA. The average division index from all animals atDay 3 or Day 30 for CD3+ cells stimulated with ConA was significantlyhigher than the division index for CD3+ cells from vehicle and pABM-SCtreated animals at pre-treatment and at necropsy (*p<0.05). See, FIG.17.

Example 11 Clinical Development

A Phase 1, open label, dose escalation study to evaluate the safety of asingle escalating dose of hABM-SC administered by endomyocardialinjection to cohorts of adults 30-60 days following initial acutemyocardial infarction has been undertaken. The primary objective of thisstudy was investigate the safety and feasibility of single escalatingdoses of hABM-SC delivered via multiple endomyocardial injections usingthe MYOSTAR™ catheter, guided by the NOGA™ or NOGA XP™ electromechanicalcardiac mapping system. A secondary objective was to investigate thepreliminary efficacy of single escalating doses of hABM-SC, measured byleft ventricular volume, dimension, myocardial infarction size andvoltage.

The study protocol provides that test subjects are to be followed for 12months with frequent monitoring for safety. Efficacy assessments are tobe performed at 90 day and six month follow-up visits. The intendedstudy population is 30 to 75 year old consenting adults with an acutemyocardial infarction (AMI) within the previous 30 days who have beensuccessfully treated with percutaneous revascularization restoring TIMIII or higher flow, with a left ventricular ejection fraction of greaterthan or equal to 30% as measured by myocardial perfusion imaging(SPECT).

Inclusion and Exclusion Criteria:

Inclusion criteria for the study comprises: (1) 30-75 years of age(inclusive); (2) 30-60 days since AMI (defined as the most recent MIcausing a doubling in cardiac-specific troponin I (cTnI) enzymeconcentrations relative to normal levels in addition to ECG changesconsistent with MI with confirmation by myocardial perfusion imaging[SPECT]); (3) successful percutaneous revascularization of restoringTIMI II or higher flow to infarcted area; (4) negative pregnancy test(serum hCG) in women of childbearing potential (within 24 hours prior todosing); (5) left ventricular ejection fraction (LVEF)>30% as measuredby myocardial perfusion imaging (SPECT); (6) cardiac enzyme tests (CPK,CPK MB, cTnI) within the normal range at baseline; (7) must beambulatory.

Exclusion Criteria for the Study Comprises:

(1) significant coronary artery stenosis that may require percutaneousor surgical revascularization within six months of enrollment; (2) leftventricular (LV) thrombus (mobile or mural); (3) high gradeatrioventricular block (AVB); (4) frequent, recurrent, sustained (>30seconds) or non-sustained ventricular tachycardia >48 hours after AMI;(5) clinically significant electrocardiographic abnormalities that mayinterfere with subject safety during the intracardiac mapping andinjection procedure; (6) atrial fibrillation with uncontrolled heartrate; (7) severe valvular disease (e.g., aortic stenosis, mitralstenosis, severe valvular insufficiency requiring valve replacement);(8) history of heart valve replacement; (9) idiopathic cardiomyopathy;(10) severe peripheral vascular disease; (11) liver enzymes (Aspartateaminotransferase [AST]/alanine aminotransferase [ALT])>3 times upperlimit of normal (ULN); (12) serum creatinine >2.0 mg/dL; (13) history ofactive cancer within the preceding three years (with exception of basalcell carcinoma); (14) previous bone marrow transplant; (15) known humanimmunodeficiency virus (HIV) infection; (16) evidence of concurrentinfection or sepsis on chest X-ray (CXR) or blood culture; (17)participation in an experimental clinical trial within 30 days prior toenrollment; (18) alcohol or recreational drug abuse within six monthsprior to enrollment; (19) major surgical procedure or major traumawithin the 14 days prior to enrollment; (20) known autoimmune disease(e.g., systemic lupus erythematosus [SLE], multiple sclerosis); (21)clinically significant elevations in prothrombin (PT) or partialthromboplastin time (PTT) relative to laboratory norms; (22)thrombocytopenia (platelet count <50,000/mm3); (23) inadequatelycontrolled diabetes mellitus type I or type II, defined as a change inanti-diabetic medication regimen within the prior 3 months orHbAlc >7.0%; (24) uncontrolled hypertension defined as systolic bloodpressure (SBP)>180 mmHg and/or diastolic blood pressure (DBP)>100 mmHg;(25) use of ionotrophic drugs>24 hours post AMI; (26) other co-morbidconditions such as hemodynamic instability, unstable arrythmias, andintubation, which, in the opinion of the principal investigator, mayplace subjects at undue risk or interfere with the objectives of thestudy; (27) any other major illness, which, in the opinion of theprincipal investigator, may interfere with the subject's ability tocomply with the protocol, compromise subject safety, or interfere withthe interpretation of the study results; and, (28) contraindication(either allergy or impaired renal function) to injection with contrastmedia for adequate CT scan evaluations.

Study Drug Dosage and Administration:

Subjects in the same cohort will be dosed no closer than three daysapart, and dosing of successive cohorts will be separated byapproximately four weeks, following review of at least three weeks ofsafety data on all subjects in the previous cohort.

Cohorts Dose Cohort 1  30 × 10⁶ cells Cohort 2 100 × 10⁶ cells Cohort 3300 × 10⁶ cells Cohort 4 300 × 10⁶ cells or MTD

On the day of dosing, after baseline evaluations are complete andimmediately following percutaneous ventricular mapping with the NOGA™ orNOGA XP™ electromechanical mapping system (Biosense Webster, DiamondBar, Calif.), multiple sequential injections of hABM-SC will bedelivered directly into the myocardium from a percutaneous, LV approachusing a MYOSTAR™ catheter.

Study Procedures:

Potential subjects will be consented and screened within 21 days priorto planned hABM-SC administration, which must occur within 30-60 daysfollowing AMI. Screening procedures to determine eligibility also willbe used as baseline values, unless hospital SOPs require additionaltests (i.e., immediately prior to catheterization). Baseline testing fortreatment efficacy is to consist of a Six Minute Walk Test, NYHAclassification, blood analysis for B-type natriuretic peptide (BNP)concentration, echocardiography, right and left cardiac catheterization,myocardial perfusion imaging (SPECT), and NOGA™ or NOGA XP™electromechanical mapping. On the day of admission, additional baselineblood testing (including pregnancy testing [serum_hCG] for femalesubjects of childbearing potential) will be done, and eligibility willbe verified. On the day of dosing (Study Day 0), subjects will undergoNOGA™ or NOGA XP™ electromechanical cardiac mapping and a MYOSTAR™catheter will be placed into the left ventricle. The dose of hABM-SCwill be administered via multiple sequential endomyocardial injectionsinto the damaged (defined by NOGA™ or NOGA XP™) myocardial tissue. Afteradministration of hABM-SC, echocardiography will be performed to detectpossible transmural perforation, and the subject will be admitteddirectly to the intensive care unit (ICU) for a minimum of twenty-fourhours of observation with continuous cardiac telemetric monitoring.Stable subjects without complications will be discharged from the ICU toa step down unit (with cardiac monitoring) and then discharged to homeno sooner than 72 hours after the dosing procedure. Subjects withcomplications will remain in the ICU under optimal medical managementuntil stable and appropriate for discharge to the step down unit. Safetyevaluations will be performed 7, 14, 21, 60 and 90 days and at six andtwelve months following administration of hABM-SC. Efficacy evaluationswill be performed at 90 days and six months after the procedure, andwill include left ventricular volume, dimension, size of myocardialinfarction and voltage, measured respectively by contrast enhancedechocardiography, myocardial reperfusion and viability analysis (SPECT),right and left cardiac catheterization (90 days only), six minute walktest, NYHA classification, and NOGA™ or NOGA XP™ electromechanicalmapping (90 days only).

Safety endpoints in the study will comprise: (1) adverse events asdetailed in the study protocol; (2) clinically significant changes frombaseline in blood or blood components including CBC, CMP, CPK/CPK MB,cTnl, PT/PTT, and HLA antibody analysis; (3) clinically significantchanges from baseline in cardiac electrical activity as assessed byelectrocardiogram (ECG) or cardiac telemetry; (4) clinically significantchanges from baseline in cardiac electrical activity as assessed by 24hour Holter monitoring; (5) perioperative myocardial perforation asassessed by echocardiogram (post procedure); and, (6) clinicallysignificant changes from baseline in physical and mental status asassessed by physical examination including a focused neurologicalexamination. If signs and symptoms consistent with cerebrovascularaccident (stroke) are observed, a neurological consult will be obtainedfor further evaluation

Efficacy Endpoints:

Efficacy endpoints to be monitored comprise: (1) end systolic and/or enddiastolic volume compared to baseline, as measured by myocardialperfusion imaging (SPECT); (2) myocardial infarction size compared tobaseline as measured by myocardial perfusion imaging (SPECT); (3) endsystolic and/or end diastolic dimensions compared to baseline asmeasured by contrast enhanced 2-D echocardiography; (4) action potentialvoltage amplitude in the area of hABM-SC injected myocardium as comparedto baseline and historical controls (provided by the core laboratory) asmeasured by NOGA™ or NOGA XP™ electromechanical mapping; (5) cardiacoutput and pressure gradients compared to baseline as determined byright and left cardiac catheterization; (6) quality of life compared tobaseline as assessed by the Six Minute Walk Test; and, (7) functionalcardiovascular disease class (NYHA functional classification scheme)compared to baseline as assessed by the physician performing scheduledphysical examinations.

Endomyocardial Delivery of hABM-SC:

hABM-SC will be delivered to the myocardium via direct catheter-guidedinjection from within the ventricular chamber. Endomyocardial deliveryof hABM-SC will be accomplished with the aid of the NOGA™ CardiacNavigation System (one of the most advanced systems for threedimensional visualization of the physical, mechanical and electricalproperties of intact myocardium in vivo; from Biosense-Webster, DiamondBar, Calif.). The actual injection will be performed with the CordisMYOSTAR™/catheter. The NOGA™ system allows for real time viewing of leftventricular heart function, detection of heart tissue damage,observation and placement of the catheter tip. Given the tenuous cardiaccondition of patients post-AMI, a relatively non-invasive deliverysystem (compared to open heart or direct intracardiac delivery), i.e.the MYOSTART™ Injection Catheter used in conjunction with the NOGA™mapping system, was selected for administration of hABM-SC.

Preliminary Results:

Preliminary results for 5 patients have been obtained. The first 3patients comprised the initial dose group (30 million cells), while thelast two patients received the second escalating dose (100 millioncells). Overall, hABM-SC was well tolerated in all patients, with sometrends to improvement in cardiac function noted in several patients.More detailed results are discussed below.

Safety Findings:

No evidence of allogeneic immune response (as measured by pre- andpost-treatment antibody profiling) was found in any patients.

Cardiac Functional Assessments:

NOGA Electromechanical Mapping: Functional mapping was performed at timeof treatment and at 90 days after cell treatment. Representativeunipolar voltage maps were obtained from the second patient in the firstdose cohort. A clear voltage deficit could be seen in the area ofinfarct (data not shown). Fifteen cell injections were performed at themargin of the infarct using unipolar voltage as a guide. At 90 daysfollow up, a clear improvement in unipolar voltage could be seen, withnear normal voltages prevailing in the infarct zone. Similar degrees ofimprovement in voltage were noted in patients 1, 3 and 4 (data notshown).

Myocardial Perfusion Imaging (SPECT):

Perfusion imaging was performed at baseline, 90 days and 6 months aftercell treatment according to previously published methods.

All images were digitally captured and analyzed. Ejection fraction,perfusion deficit size, and ventricular volumes were derived from thisanalysis, under basal and adenosine-stress conditions, along with a 24hour-washout rescan. Results for each patient at each time point arediscussed below.

Perfusion Deficit:

In general, perfusion deficit sizes, which are thought to representoverall infarct sizes, either decreased or remained unchanged over thesix months of follow up for treated patients. Two patients demonstratedreductions in deficit deemed “clinically significant” meaning thedeficits resolved to less than 4-5% of the total ventricular wall. Inboth of these cases, the areas of improvement corresponded to areas ofvoltage improvement as measured by NOGA mapping. Although NOGA mappingis considered investigational, this data supports validity of thehypothesis that unipolar voltage may be a surrogate for infarct sizemeasurement.

Ejection Fraction (EF):

In general, ejection fraction in study patients either improved orremained relatively unchanged. One patient experienced a significantdrop in overall EF (63% to 50% over six months), but this patientexperienced a serious adverse event during the course of cell treatmentwhich renders it questionable whether or not a complete dose of cellswas actually administered to the endomyocardium. Two patientsdemonstrated increases in EF well above the expected for this patientgroup. The lack of placebo controls precludes any conclusions as to themechanism of this improvement.

End-Diastolic Volume (EDV):

EDV was measure at baseline and at 90 days and 6 months followingtreatment. In general, EDV remained unchanged in all patients over the 6month follow up period, suggesting no significant remodeling occurred inthese patients.

FIG. 18 shows the changes in cardiac fixed perfusion deficit size inthree patients by comparison of a baseline (BL) measurements withmeasurements obtained 90 days post-treatment with hABM-SC. FIG. 19 showsthe changes in cardiac ejection fractions measured in three patients bycomparison of a baseline (BL) measurements with measurements obtained 90days post-treatment with hABM-SC.

Example 12 Human ABM-SC and Compositions Derived Thereby for theProduction of Red Blood Cells In Vitro

It is well known that the bone marrow microenvironment provides therequisite combination of matrix molecules, growth factors and cytokinesnecessary to support and modulate hematopoiesis (Dexter at al. 1981).Most, if not all, of the trophic factors known to drive hematopoieticcell self-renewal and lineage restricted differentiation derive from themesenchymal support cells (Quesenberry et al. 1985). Roecklein andTorok-Storb (1995) showed that even within a relatively pure populationof these cells, sub-populations can be isolated that differentiallysupport hematopoiesis. Unlike the immortalized clones described in theseprevious publications, the hABM-SC utilized as described hereinrepresent a pure population of CD45 negative, CD90/CD49c co-positivenon-hematopoietic support cells that secrete many factors important forinducing and maintaining erythropoiesis including, but not limited to,IL-6 (Ullrich et al. 1989), LIF (Cory et al. 1991), SDF-1 (Hodohara etal. 2000), SCF (Dai et al. 1991), Activin-A (Shao et al. 1992), VEGF andIGF-II (Miharada et al. 2006) (FIG. 20).

To generate red blood cells from a starting population of hematopoieticprecursors (e.g. embryonic stem cells (ES), hematopoietic stem cells(HSC), cord blood cells (CBC) or committed erythroblast precursors(BFU-E)), human ABM-SC and/or compositions produced by such cells can beutilized to induce, enhance, and/or maintain erythropoiesis bydelivering a “cocktail” of erythropoietic factors necessary for, or tosupplement, growth and differentiation of hematopoietic precursors intoerythroblasts. See, FIG. 20.

Example 13 Production, Isolation, Purification, and Packaging ofCell-Derived Compositions and Trophic Factors

A two-step, downstream bioprocess has been developed to manufacture,collect and purify compositions such as secreted growth factors,cytokines, soluble receptors and other macromolecules produced by humanABM-SC and exABM-SC. This cocktail of secreted cell compositions,produced as such in the stoichiometric ratios created by the cells, hastremendous potential as a pro-regenerative therapeutic, cell culturereagent and/or research tool for studying in vitro cell and tissueregeneration. Such compositions can also be used as an alternative tothe cells themselves to support the growth and lineage-appropriatedifferentiation of starting erythroid progenitor cell populations insuspension cultures.

Production of Sera-Free Conditioned Media

Cryopreserved human ABM-SC (Lot no. P25-T2S1F1-5) are thawed andre-suspended in one liter of Advanced DMEM (GIBCO, catalog #12491-015,lot 284174 (Invitrogen Corp., Carlsbad, Calif., USA)) supplemented with4 mM L-glutamine (HYCLONE Laboratories Inc., Logan, Utah, USA catalog#SH30255.01).

Cells are seeded in a Corning® CellBind® polystyrene CellSTACK® tenchamber (catalog number 3312, (Corning Inc., NY, USA)) at a density of20,000 to 25,000 cells per cm². One port of the CellSTACK® ten chamberunit is fitted with a CellSTACK® Culture chamber filling accessory(Corning® Catalog number 3333, (Corning Inc., NY, USA)) while the otherport is fitted with a CellSTACK® Culture chamber filling accessory 37mm, 0.1 μM filter (Corning® Catalog number 3284, (Corning Inc., NY,USA)).

Cultures are placed in a 37° C.±1° C. incubator and aerated with a bloodgas mixture (5±0.25% CO₂, 4±0.25% O₂, balance Nitrogen (GTS, Allentown,Pa.)) for 5±0.5 hrs. After 24±2 hrs post seeding, the media is removed,replaced with 1 liter of fresh media and aerated as previouslydescribed. Approximately, 72±2 hours later the sera-free conditionedmedia is aseptically removed from the CellSTACK® ten chamber unit withina biological safety cabinet and transferred to a one liter PETG bottle.The sera free conditioned media is subsequently processed by tangentialflow filtration.

Isolation and Purification of Sera-Free Conditioned Media

Tangential flow filtration (TFF) is performed on a reservoir of serafree conditioned media, recovered from a CellSTACK® ten chamber unit, asdescribed above. A polysulfone hollow fiber with a molecular weightcut-off of 100 kilodaltons (kD) (Catalog number M1ABS-360-01P (SpectrumLaboratories, Inc., Rancho Dominguez, Calif., USA)) is employed. Thereservoir of sera free conditioned (the retentate) is re-circulatedthrough the lumen of the hollow fiber tangential to the face of thelumen. Molecules with a molecular weight of 100 kD or less pass throughthe lumen into a 2 liter PETG bottle; this fraction is called thepermeate or filtrate. The retentate is continually re-circulated untilthe volume is reduced to approximately less than 50 mL. The retentate issubsequently discarded and the permeate is retained for furtherprocessing. The resulting permeate (approximately 1 liter) is a clear,sera-free solution containing small molecular weight molecules free ofcellular debris and larger macromolecules, herein referred to asFraction #1.

Fraction #1 is subsequently subjected to additional TFF using apolysulfone hollow fiber with a molecular weight cut off of 10kilodaltons (kD) (Catalog number M11S-360-01P (Spectrum Laboratories,Inc., Rancho Dominguez, Calif., USA)). Fraction #1 is subsequently usedas the retentate and re-circulated through the lumen of the hollowfiber, tangential to the face of the lumen. Smaller molecules ≦10 kD(i.e. ammonia, lactic acid etc.) are allowed to pass through the lumen.After the volume of the retentate is reduced to 100 mL, diafiltration ofthe solution is begun. One liter of alpha-MEM without phenol red(HYCLONE, catalog number RR11236.01 (HYCLONE Laboratories Inc., Logan,Utah, USA)) is added to the retentate reservoir at the same rate thatthe permeate is pumped out; thus maintaining the volume of the reservoirconstant. The resulting retentate contains small only small moleculesranging in molecular weight from 10 kD to 100 kD; herein referred to asFraction #2.

Fraction #2 can be further processed by subjecting it to additional TFFusing a polysulfone hollow fiber with a molecular weight cut off of 50kilodaltons (kD) (Catalog number M15S-360-01P (Spectrum Laboratories,Inc., Rancho Dominguez, Calif., USA)). Fraction #2 is thus re-circulatedthrough the lumen of the hollow fiber, tangential to the face of thelumen. Smaller molecules ≦50 kD are passed through the lumen. Bothprocessing streams are retained as product. The resultingpermeate/filtrate is composed primarily of molecules 10 kD to 50 kD(Fraction #3), while the retentate comprises macromolecules in the rangeof 50 kD to 100 kD (Fraction #4).

Each of the resulting fractions is frozen in 60 mL PETG bottles (Catalognumber 2019-0060, Nalgene Nunc International Rochester N.Y.).

Such Isolated protein fractions can subsequently be subjected to furtheraseptic downstream processing and packaging, wherein such compositionscan be dialyzed, lyophilized, and reconstituted into a dry,biocompatible matrix, such as LYOSPHERES™ (manufactured by BIOLYPH™,Hopkins, Minn., USA).

Example 14 Isolation, Cryopreservation, and Expansion of CD34+ CordBlood Cells (CBC)

Large scale production of lineage-committed erythroid cells (CFU-E orReticulocytes) can be manufactured from a starting population of stemcells or erythroblast precursors (e.g. cord blood cells, embryonic stemcells, hematopoietic stem cells and BFU-E) employing the methodsdescribed below.

Umbilical cord blood from healthy full-term newborns is collected inheparinized blood collection bags. A clean nucleated cell preparation ismade by adding ammonium chloride lysis solution to cord blood, thencentrifuging the mixture at 300×g for 15 minutes at room temperature.The supernatant is aspirated from the cell pellet, and the cell pelletis washed in BSSD with 5% human serum albumin (wash solution). The cellsare centrifuged again at 300×g for 15 minutes at room temperature andthe wash solution is removed from the cell pellet by aspiration. CD34+cells are separated by magnetic cell sorting using MASC LS-columns(MACS®; Miltenyi Biotech, Gladbach, Germany) using establishedprotocols. The CD34+ CBCs are subsequently re-suspended in CSM-55 atapproximately 2 million cells/mL and cryopreserved using acontrolled-rate freezer.

BSSD (Balanced Salt Solution with 4.5% Dextrose) is prepared as follows:

To Balanced Salt Solution, Sterile Irrigating Solution (BSS; Baxter,Deerfield, Ill., USA) add 450±0.5 grams Dextrose (EMD Life Sciences,Gibbstown N.J. USA), QS to a final volume of 10.0 Liters with BSS.

CSM-55 (Cryogenic Storage Media 5% DMSO, 5% HSA) is Prepared as Follows:

In a 2 liter bottle combine 1.4 liters of BSSD with 400 mLs of 25% HSA(25% solution human serum albumin from ZLB Behring, Ill., USA) and 200mLs of 50% DMSO (50% dimethyl sulfoxide from Edwards Lifesciences IrvineCalif. USA).

Wash Solution is Prepared with 400 mLs of BSSD Plus 100 mLs of 25% HSA.

CD34+ CBC Expansion in Suspension Cultures

The cells are subsequently re-suspended in StemSpan® H300 (StemCellTechnology) supplemented with 1.0 U/mL recombinant human EPO(R&DSystems, Cat #287-TC), 10 LYOSPHERES™/L, and inoculated into adisposable HYCLONE™ perfusion BIOPROCESS CONTAINERT™ (bioreactor) orequivalent, at a cell concentration of 1.0×10⁶/mL. Cultures aremaintained at 37° C. with 5% CO₂, 4% O₂, and balanced with Nitrogen, for3 weeks using continuous flow of fresh culture media. On day 14,cultures are supplemented with the glucocorticoid antagonistMifepristone to accelerate enucleation, as described by Miharada et al.2006. Continuous flow of fresh culture media is maintained at a fixedrate under these conditions until harvest on day 21.

CBC Expansion on a Human ABM-SC Feeder Layer

Cryopreserved human ABM-SC are thawed and re-suspended in Advanced RPMIMedia 1640 (INVITROGENT™) supplemented with 1.0 U/mL recombinant humanEPO(R&D Systems, Cat #287-TC), 4 mM L-Glutamine, 10% lot selected,gamma-irradiated fetal bovine serum (Hyclone), and seeded at a densityof 10,000 cells/cm² in 40 layer cell culture factories (Corning) andmaintained at 37° C. under 5% CO₂, 4% O₂, and balanced with Nitrogen at37° C. On day 5, one-half volume of spent media is removed from thecultures and replenished by adding back one-half volume of fresh mediaalong with 1.0×10⁶ CBC/mL. Discontinuous flow (on-off-on) of freshculture media is subsequently engaged to enable the media conditions tocycle between fresh (on) to conditioned (off), and back to fresh mediaagain (on). On day 14, cultures are supplemented with the glucocorticoidantagonist Mifepristone to accelerate enucleation, as described byabove. Co-cultures are maintained under these conditions until harveston day 21.

Example 15 ABM-SC Secrete Scavenger Receptors and Antagonists And ReduceTumor Necrosis Factor-Alpha Levels in a Dose Dependent Manner

Background:

Embodiments of the present invention include methods and compositionsfor treating, reducing, or preventing adverse immune activity (such asinflammation or autoimmune activity) in a subject by deliveringtherapeutically effective amounts of exABM-SC or compositions producedby exABM-SC. Embodiments of the invention include utilization ofexABM-SC, or compositions produced thereby, relying on the naturallyoccurring or basal level production of secreted compositions in vitro.Alternatively, embodiments of the invention also include utilization ofexABM-SC, or compositions produced thereby, by manipulating the exABM-SCto modulate (up- or down-regulate) the quantity and kind of compositionsproduced (for example, by administration of pro-inflammatory factorssuch as TNF-alpha).

For example, it has now been found that exABM-SC produce at least onescavenger receptor for the cytokine Tumor Necrosis Factor-alpha (TNF-α),and at least one antagonist of the Interleukin-1 Receptor (IL-1R), andat least one binding protein (antagonist) of cytokine Interleukin-18(IL-18). Accordingly, embodiments of the invention include methods andcompositions for use and administration of stable cell populations (suchas exABM-SC) that consistently secrete therapeutically useful proteinsin their native form.

The term “stable cell population” as used herein means an isolated, invitro cultured, cell population that when introduced into a livingmammalian organism (such as a mouse, rat, human, dog, cow, etc.) doesnot result in detectable production of cells which have differentiatedinto a new cell type or cell types (such as a neuron(s),cardiomyocyte(s), osteocyte(s), hepatocyte(s), etc.) and wherein thecells in the cell population continue to secrete, or maintain theability to secrete or the ability to be induced to secrete, detectablelevels of at least one therapeutically useful composition (such assoluble TNF-alpha receptor, IL-1R antagonists, IL-18 antagonists,compositions shown in Table 1A, 1B and 1C, etc.).

For purposes of the present invention, “scavenger receptor” is intendedto mean any soluble or secreted receptor (whether membrane bound or freein the extracellular milieu) capable of binding to and neutralizing itscognate ligand.

In addition to the pro-inflammatory factors listed above, in view of thepresent disclosure it is also understood that cell populations of thepresent invention may be treated with any number, variety, combination,and/or varying concentrations of factors now known or subsequentlydiscovered or identified in order to manipulate the concentration andkind of compositions produced by cell populations of the presentinvention. For example, the cell populations of the invention maypreferably be treated with factors such as: IL-1alpha, IL-1beta, IL-2,IL-12, IL-15, IL-18, IL-23, TNF-alpha, TNF-beta, and Leptin. This brieflist of preferred factors, however, is not intended nor should it beconstrued as limiting with respect to the number of differentcompositions that can be used to treat cell populations of the presentinvention, nor are these compositions limited to proteins, as is it isalso appreciated that many other types of compounds could also be usedto manipulate the cell populations of the present invention (including,by way of brief examples, other biological macromolecules such asnucleic acids, lipids, carbohydrates, etc. and small molecules andchemicals such as dimethylsulfoxide (DMSO) and nitrous oxide (NO), etc).

Methods:

Production of serum-free conditioned media was produced as describedbelow for use in enzyme-linked immunosorbant assays (ELISA) (alsodescribed below). Cryopreserved human exABM-SC (Lot # MFG-05-15; at ˜43population doublings) were thawed and re-suspended in Advanced DMEM(GIBCO™; Catalog #12491-015, Lot #1216032 (Invitrogen Corp., Carlsbad,Calif. USA)) supplemented with 4 mM L-glutamine (Catalog #SH30034.01.Lot #134-7944, (HYCLONE© Laboratories Inc., Logan, Utah, USA)) with andwithout 10 ng/mL TNF-α. Cell suspensions were then seeded in T-225 cm²CELLBIND™ (Corning Inc., NY, USA) culture flasks (culture surfacestreated with a patented microwave plasma process; see, U.S. Pat. No.6,617,152) (n=3) at 10,000, 20,000, 40,000 cells/cm² in 36 mL of media(n=3 per condition). Heat-inactivated cells seeded at 40,000 cells/cm²were used as a negative control. Cells were heat-inactivated bytransferring an aliquot to a sterile tube and incubating it for ˜40minutes in a 70° C. heat block containing water (for efficient heattransfer). Cultures were placed in a 37° C. humidified trigas incubator(4% O₂, 5% CO₂, balanced with nitrogen) for approximately 24 hours.Cultures were then re-fed with fresh media on same day to removenon-adherent debris and returned to the incubator. On day 3, cellculture media was concentrated using 20 mL CENTRICON™ PLUS-20Centrifugal Filter Units (Millipore Corp., Billerica, Mass., USA), asper manufacturer's instructions. Briefly, concentrators were centrifugedfor 45 minutes at 1140×G. Concentrated supernatants (100× finalconcentration) were transferred to clean 2 mL cryovials and stored at−80° C. until later use.

To determine the levels of certain secreted proteins produced from thehuman ABM-SC in these adherent cultures, enzyme-linked immunosorbantassays (ELISA) were performed on day 3, 100× concentrated, conditionedcell culture supernatants collected as described above. On the day ofassay, supernatants were thawed and equilibrated to room temperaturebefore use. ELISA analysis was performed to detect TNF-α, soluble TNF-RI(sTNF-RI), soluble TNF-RII (sTNF-RII), IL-1 receptor antagonist (IL-IRA)and IL-2 receptor alpha (conducted as per manufacturer's instructions;all kits were purchased from R&D Systems, Inc. (Minneapolis, Minn.,USA)).

The results demonstrate that therapeutically relevant levels of secretedscavenger receptors (e.g. sTNF-RI) and receptor antagonists (e.g.IL-IRA) are produced by these adherent cultures and that these levelscan be controlled by adjusting cell concentration or dose (FIG. 21-23).Importantly, these data also demonstrate that the cells respond to theinflammatory milieu in which they are placed. For example, followingtreatment with the potent inflammatory cytokine TNF-alpha, the cellsup-regulate secretion of sTNF-RII (FIG. 22B) and IL-IRA (FIG. 23).Interestingly, in these sample cultures, the levels of TNF-alpha weresignificantly reduced with each increase in cell seeding density (FIG.21), suggesting that the TNF-alpha itself was sequestered in some way byeither the ABM-SCs or factors that they secrete.

It is well established that both sTNF-RI and sTNF-RII can bind andneutralize the biological activity of TNF-alpha. Since the measurablelevels of both forms of the TNF receptor, as well as TNF-alpha itself,are each reduced significantly with each increase in cell seedingdensity, it is likely that the ABM-SC derived sTNF-RI and sTNF-RII arebinding to and masking TNF-alpha in this assay system.

Of the soluble receptors and receptor antagonists measured, detectablelevels were not seen in cultures containing heat-inactivated cells only.Statistical comparisons between assay conditions were determined byone-way ANOVA.

Example 16 Osteogenesis Induction Assay: Human ABM-SC Cells do notExhibit a Bone Differentiation Characteristic In Vitro when CellPopulations Expanded Beyond Approximately 25 Population Doublings areExposed to Standard Osteoinductive Conditions or when Cell PopulationsExpanded Beyond Approximately 30 Population Doublings are Exposed toEnhanced Osteoinductive Conditions

Methods:

Human ABM-SC and exABM-SC were seeded at 3100 cells/cm² in 6-wellculture dishes (Corning, Catalog #3516) with 2.4 mL Mesenchymal StemCell Basal Medium (MSCBM™; Lonza, Catalog #PT-3238) supplemented withMSCGM™ SingleQuot Kit (Lonza, Catalog #PT-4105) per well, hereafterreferred to as Mesenchymal Stem Cell Growth Medium (MSCGM™).Approximately four hours later, the MSCGM™ was changed to theappropriate test conditions. Negative control wells were those re-fedwith either MSCGM™ alone, or MSCGM™ supplemented with 5 ng/mLrecombinant mouse Noggin/Fc Chimer (R&D Systems, Catalog #719-NG). Thetest wells were those treated with either Osteogenesis Induction Medium(OIM; Lonza Catalog #PT-3924 and #PT-4120) alone (standardosteoinductive conditions) or OIM supplemented with 5 ng/mL recombinantmouse Noggin/Fc Chimer (enhanced osteoinductive conditions). Cultureswere then maintained in a humidified CO₂ incubator at 37° C. and re-fedwith fresh medium every 3-4 days for 2 weeks. After 14 days, cultureswere processed for calcium determination using the Calcium Liquicolorkit (Stanbio, Catalog #0150-250), as per manufacturer's instructions.Plates were read at 550 nm using a SpectraMax Plus³⁸⁴ microplate reader.

Results:

Human ABM-SC and exABM-SC derived from research lot #MCB 109 werecultured under standard osteoinductive conditions (OIM only) or underenhanced osteoinductive conditions (OIM and the morphogen Noggin;OIM+Noggin). Negative control cultures were maintained in either growthmedia alone (MSCGM™) or MSCGM™ supplemented with Noggin (MSCGM™+Noggin).

ABM-SC at about 16 population doublings exhibited a calcium depositionincrease of approximately 6-fold when the OIM media was supplementedwith Noggin (i.e., ABM-SC at about 16 population doublings deposited ˜5micrograms calcium/well under OIM conditions and ˜30 micrograms/wellunder OIM+Noggin conditions). ABM-SC lost the capacity to depositdetectable levels of calcium beyond about 16 population doublings understandard OIM conditions, however, this could be reversed bysupplementing with Noggin (i.e., exABM-SC at about 25 populationdoublings deposited no detectable calcium under OIM conditions whereasthese same cells deposited ˜5 micrograms calcium/well under OIM+Nogginconditions). In contrast, beyond about 30 population doublings (e.g., atabout 35 and 43 populations doublings) exABM-SC did not depositdetectable levels of calcium under any of the conditions tested(standard or enhanced OIM).

Example 17 Expression of IL-1 Receptor Antagonist (IL-1RA) and IL-18Binding Protein (IL-18BP) by ABM-SC

Methods:

Human ABM-SC which had undergone about 43 cell population doublings (lot# P17-T2S1F1-5) were thawed and seeded in AFG growth medium supplementedwith Brefeldin A at 3 micrograms/mL (1×) and placed in a humidified 5%CO₂ incubator at 37° C. for 24 hours. Cultured cells were then removedfrom the culture flasks using porcine trypsin, washed and prepared forflow cytometry, as per CALTAG FIX & PERM® staining protocol (CALTAGLABORATORIES; now part of Invitrogen Corp. (Carlsbad, Calif., USA).Cells were stained with either FITC conjugated mouse anti-human IL-1Receptor Antagonist (IL-1RA; eBioscience, Catalog #11-7015, clone CRM17)antibody neat or unlabeled rabbit anti-IL-18 Binding Protein (IL-18BP;Epitomics, Catalog #1893-1, clone EP1088Y) at a 1:10 dilution, both for45 minutes at room temperature. FITC-rabbit FITC-labeled goatanti-rabbit antibody was then used to detect the IL-18BP. Isotypematched controls were included as a negative control (Beckman Coulter).

Results:

Human exABM-SC express basal levels of IL-1 receptor antagonist (IL-1RA;FIG. 24A) and IL-18 binding protein (IL-18BP; FIG. 24B) even in theabsence of an inflammatory signal such as TNF-alpha.

Example 18 Human ABM-SC Reduce Expression of TNF-Alpha and IL-13 WhileSimultaneously Increasing Expression of IL-2

Methods:

Human peripheral blood mononuclear cells (PBMC) were co-cultured inRPMI-1640 containing 5% Human Sera Albumin, 10 mM HEPES, 2 mM glutamine,0.05 mM 2-mercaptoethanol, 100 U/mL penicillin, and 100 microg/mLstreptomycin, in a 24 well plate with either 1) Mitomycin-C treated PBMCfrom same donor (Responder+Self) or 2) Mitomycin-C treated PBMC derivedfrom a different donor (Responder+Stimulator). PBMC from each sourcewere each seeded at 4×10⁵ cells/well. For each condition, cultures weresupplemented with or without human ABM-SC at a seeding density of 40,000cells/well. Cultures were maintained in a humidified 5% CO₂ incubator at37° C. for 7 days to condition the media. Conditioned cell culturesupernatants were collected and analyzed for the presence of the variouscytokines using the SEARCHLIGHT™ 9-Plex assay (Pierce Protein ResearchProducts, Thermo Fisher Scientific Inc., Rockford, Ill.). Statisticalanalysis was performed by one-way ANOVA (analysis of variance).

Results:

Co-culture of allogeneic PBMC (Responders+Stimulators) resulted in amarked increase in the levels of TNF-alpha and IL-13, as would beexpected for a mixed PBMC reaction. When challenged with human ABM-SC,however, both IL-13 and TNF-alpha were significantly reduced (P<0.001),suggesting that ABM-SC could be utilized therapeutically to treatchronic inflammatory disorders or graft rejection by reducing focal orserum levels of inflammatory mediators. See, FIGS. 25A, B, and C.

Notably, ABM-SC induced elevated expression of IL-2 in both autologous(Responders+Self) and allogeneic (Responders+Stimulators) mixed PBMCcultures (P<0.001) while simultaneously suppressing PBMC proliferation.While this result appears somewhat paradoxical given the importance ofIL-2 in promoting T cell proliferation, recently it has been shown inmice that disruption of the IL-2 pathway results in lymphoid hyperplasiaand autoimmunity rather than immune deficiency, suggesting that themajor physiological role of IL-2 may be to limit or regulate, ratherthan enhance T cell responses (Nelson, “IL-2, Regulatory T-Cells, andTolerance,” Jour. Immunol. 172: 3983-3988 (2004)). Additionally, it isnow known that IL-2 is also critical for promoting self-tolerance bysuppressing T cell responses in vivo and that the mechanism by whichthis occurs is through the expansion and maturation of CD4+/CD25+regulatory T cells. It is, therefore, contemplated that ABM-SC could beemployed therapeutically to induce T-cell tolerance by indirectlysupporting the maturation of T regulatory cells through the inducedup-regulation of IL-2.

Example 19 Human ABM-SC Inhibit Mitogen-Induced Peripheral BloodMononuclear Cell Proliferation

Methods:

Human adult bone-marrow derived somatic cells (ABM-SC) were cultured invitro for 96 hours in a humidified incubator under 5% CO₂ then passagedonto 96-well round bottom plates at a concentration of 25,000 viablecells/mL in RPMI-complete media (HYCLONE™). Human peripheral bloodmononuclear cells (PBMC) were cultured either separately at 250,000cells/mL in RPMI-complete media, or with ABM-SC Lots RECB801(sub-cultured to about 19 population doublings) or RECB906 (sub-culturedto about 43 population doublings). To stimulate PBMC proliferation,cultures were inoculated with 2.5 microg/mL phytohaemagglutinin (SigmaChemical Co.). After 56 hours in culture, cells were pulsed withThymidine-[Methyl-3H] (Perking Elmer, lmicroCi/well). Cells wereharvested at 72 hours using a Filtermaster harvester onto glass filters.Filters were read in Omnifilter platers using an NXT TopCountScintillation counter. Human mesenchymal stem cells were included as apositive control. (Human mesenchymal stem cells were obtained fromCambrex Research Bioproducts; now owned by Lonza Group, Ltd, Basel,Switzerland). Statistical analysis was performed by one-way ANOVA(analysis of variance).

Results:

PBMC-induced proliferation was significantly reduced when challengedwith either lot of ABM-SC (P<0.001). See, FIG. 26. Mesenchymal stemcells (MSC) were included as a positive control. These data suggest thatABM-SC not only inhibit mitogen-induced proliferation of the total PBMCpreparation, but that the presence of ABM-SC in this assay system doesnot induce proliferation of various cell subpopulations within thepreparation (e.g., monocytes, granulocytes, lymophocytes).

REFERENCES

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1-94. (canceled)
 95. A biocompatible matrix comprising conditioned cellculture media derived from an isolated population of bone marrow-derivedself-renewing colony-forming somatic cells (CF-SC), wherein the CF-SC donot have multipotent differentiation capacity, wherein the CF-SC have anormal karyotype, wherein the CF-SC are non-immortalized, and whereinthe CF-SC express CD13, CD44, CD49c, CD90, HLA Class-1 and β (beta)2-Microglobulin, and wherein the CF-SC do not express CD10, CD34, CD45,CD62L, or CD106.
 96. The biocompatible matrix of claim 95, wherein theCF-SC further express one or more molecules selected from the groupconsisting of CD29, CD59, CD147, CD166 and telomerase, and wherein theCF-SC further do not express one or more molecules selected from thegroup consisting of CD11c, CD14, CD33, CD62P, CD80, STRO-1,HLA-Class-II, CD178, p53 and p21.
 97. The biocompatible matrix of claim95, wherein the CF-SC cells were obtained from bone marrow by a methodcomprising: (a) incubating bone marrow cells under a low oxygencondition such that the bone marrow cells when allowed to adhere to atissue culture-treated surface produce adherent colony forming units;and, (b) passaging cells in the adherent colony forming units at lowcell seeding densities.
 98. The biocompatible matrix of claim 97,wherein the low oxygen condition comprises between about 1 to 10%oxygen.
 99. The biocompatible matrix of claim 97, wherein the low cellseeding density is less than about 2500 cells/cm².
 100. Thebiocompatible matrix of claim 95, wherein the matrix is biodegradable.101. The biocompatible matrix of claim 100, wherein the matrix comprisescollagen, fibrin, polyglycolic acid (PGA), or combinations thereof. 102.The biocompatible matrix of claim 101, wherein the matrix comprises PGA.103. The biocompatible matrix of claim 95, wherein the conditioned cellculture media is serum free.
 104. The biocompatible matrix of claim 103,further comprising at least one pharmaceutical compound.
 105. Thebiocompatible matrix of claim 104, wherein the at least onepharmaceutical compound is selected from the group consisting ofanti-inflammatories, antibiotics, vitamins, minerals, extracellularmatrix proteins, blood plasma coagulation proteins, antibodies, growthfactors, chemokines, cytokines, lipids and nucleic acids.
 106. A methodof preparing a medical device, comprising: contacting conditioned cellculture media derived from bone marrow-derived self-renewingcolony-forming somatic cells (CF-SC) with a biocompatible matrix to forma medical device, wherein the CF-SC do not have multipotentdifferentiation capacity, wherein the CF-SC have a normal karyotype,wherein the CF-SC are non-immortalized, and wherein the CF-SC expressCD13, CD44, CD49c, CD90, HLA Class-1 and β (beta) 2-Microglobulin, andwherein the CF-SC do not express CD10, CD34, CD45, CD62L, or CD106. 107.The method of claim 106, wherein the conditioned cell culture media isderived from CP-SC cells cultured under a low oxygen conditioncomprising between about 1 to 10% oxygen.
 108. The method of claim 106,wherein the matrix comprises collagen, fibrin, polyglycolic acid (PGA),or combinations thereof.
 109. The method of claim 108, wherein thematrix comprises PGA.
 110. A method of repairing, treating, or promotingregeneration of damaged tissue, comprising contacting a subject in needthereof with a therapeutically effective amount of the biocompatiblematrix of claim
 95. 111. The method of claim 110, wherein the matrixfurther comprises at least one pharmaceutical compound.
 112. The methodof claim 110, wherein the tissue damage was caused by an immune relateddisorder, inflammation, ischemia, traumatic injury, or infection. 113.The method of claim 110, wherein the damaged tissue is skin, bone,connective tissue, or cartilage.
 114. The method of claim 113, whereinthe connective tissue comprises tendon or ligament.
 115. The method ofclaim 110, wherein the tissue damage is a diabetic ulcer.